Extended Dicer Substrate Agents and Methods for the Specific Inhibition of Gene Expression

ABSTRACT

The invention provides compositions and methods for reducing expression of a target gene in a cell, involving contacting a cell with an isolated double stranded nucleic acid (dsNA) in an amount effective to reduce expression of a target gene in a cell. The dsNAs of the invention possess a pattern of deoxyribonucleotides (in most embodiments, the pattern comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair) designed to direct the site of Dicer enzyme cleavage within the dsNA molecule. Deoxyribonucleotides of the dsNA molecules of the invention are located within a region of the dsNA that can be excised via Dicer cleavage to generate an active siRNA agent that no longer contains the deoxyribonucleotide pattern (e.g., deoxyribonucleotide-deoxyribonucleotide base pairs). Such DNA-extended Dicer-substrate siRNAs (DsiRNAs) were demonstrated to be more effective RNA inhibitory agents than corresponding double stranded RNA-extended DsiRNAs. DsiRNA agents were also found to tolerate guide strand mismatches.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of application Ser. No.13/971,507 filed Aug. 20, 2013, which claims priority to applicationSer. No. 12/642,371 filed Dec. 18, 2009, now U.S. Pat. No. 8,513,207issued Aug. 20, 2013, which is related to and claims priority under 35U.S.C. §119(e) to the following applications: U.S. provisional patentapplication No. 61/138,946, filed Dec. 18, 2008; U.S. provisional patentapplication No. 61/166,227, filed Apr. 2, 2009; U.S. provisional patentapplication No. 61/173,505, filed Apr. 28, 2009; U.S. provisional patentapplication No. 61/173,514, filed Apr. 28, 2009; U.S. provisional patentapplication No. 61/173,521, filed Apr. 28, 2009; U.S. provisional patentapplication No. 61/173,525, filed Apr. 28, 2009; U.S. provisional patentapplication No. 61/173,532, filed Apr. 28, 2009; U.S. provisional patentapplication No. 61/173,538, filed Apr. 28, 2009; U.S. provisional patentapplication No. 61/173,544, filed Apr. 28, 2009; U.S. provisional patentapplication No. 61/173,549, filed Apr. 28, 2009; U.S. provisional patentapplication No. 61/173,554, filed Apr. 28, 2009; U.S. provisional patentapplication No. 61/173,556, filed Apr. 28, 2009; U.S. provisional patentapplication No. 61/173,558, filed Apr. 28, 2009; and U.S. provisionalpatent application No. 61/173,563, filed Apr. 28, 2009. The entireteachings of the above applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Double-stranded RNA (dsRNA) agents possessing strand lengths of 25 to 35nucleotides have been described as effective inhibitors of target geneexpression in mammalian cells (Rossi et al., U.S. Patent PublicationNos. 2005/0244858 and 2005/0277610). dsRNA agents of such length arebelieved to be processed by the Dicer enzyme of the RNA interference(RNAi) pathway, leading such agents to be termed “Dicer substrate siRNA”(“DsiRNA”) agents. Certain modified structures of DsiRNA agents werepreviously described (Rossi et al., U.S. Patent Publication No.2007/0265220).

While robust, sequence-specific target gene silencing efficacy has beenidentified for 25-35 nucleotide length dsRNA agents, a need exists forimproved design of such agents, including design of DsiRNA agentspossessing enhanced in vitro and in vivo efficacy.

BRIEF SUMMARY OF THE INVENTION

The present invention is based, at least in part, upon the surprisingdiscovery that double stranded nucleic acid agents having strand lengthsin the range of 27-39 nucleotides in length that possess base paireddeoxyribonucleotides either at or near the 3′ terminus of the sensestrand/5′ terminus of the antisense strand or at or near the 5′ terminusof the sense strand/3′ terminus of the antisense strand are effectiveRNA interference agents. Indeed, the instant invention relates to thedemonstration that inclusion of base paired deoxyribonucleotides withina region of a Dicer substrate siRNA (“DsiRNAs”) that is excised from aresultant active siRNA via Dicer enzyme cleavage, results in aneffective inhibitory agent. Inclusion of one or more base paireddeoxyribonucleotides within this region of a DsiRNA can impart certainadvantages to such a modified DsiRNA molecule, including, e.g., enhancedefficacy (including enhanced potency and/or improved duration ofeffect), display of a recognition domain for DNA-binding molecules, andother attributes associated with a DNA:DNA duplex region. Indeed, suchdouble stranded DNA:DNA-extended DsiRNA agents were demonstrated topossess enhanced efficacy, especially including improved potency,relative to corresponding double stranded RNA:DNA- or RNA:RNA-extendedDsiRNA agents.

Among the advantages of the instant invention, the surprising discoverythat DNA-extended DsiRNA agents do not exhibit decreased efficacy asduplex length increases allows for the generation of DsiRNAs that remaineffective RNA inhibitory agents while providing greater spacing for,e.g., attachment of DsiRNAs to additional functional groups,inclusion/patterning of stabilizing modifications (e.g., PS-NA moieties)or other forms of modifications capable of adding further functionalityand/or enhancing, e.g., pharmacokinetics, pharmacodynamics orbiodistribution of such agents, as compared to dsRNA agents ofcorresponding length that do not contain such double strandedDNA-extended domains. The effect of such dsDNA-extension regions appearsnot to result from a stabilizing activity inherent in dsDNA regions, butrather appears to be attributable to the ability of specificallylocalized deoxyribonucleotide residues (either located 3′ of theprojected Dicer cleavage site of the first strand and correspondingly 5′of the projected Dicer cleavage site of the second strand or located 5′of the projected Dicer cleavage site of the first strand andcorrespondingly 3′ of the projected Dicer cleavage site of the secondstrand) to direct Dicer cleavage such that a preferred cleavage productand/or population of cleavage products is generated and/or is made moreprevalent.

Thus, in certain aspects, the instant invention allows for design of RNAinhibitory agents possessing enhanced efficacies at greater length (viamore precise direction of the location of Dicer cleavage events) thanpreviously described RNA inhibitory agents, thereby allowing forgeneration of dsRNA-containing agents possessing enhanced efficacy,delivery, pharmacokinetic, pharmacodynamic and biodistributionattributes, as well as improved ability, e.g., to be successfullyformulated, to be attached to an active drug molecule and/or payload, tobe attached to another active nucleic acid molecule, to be attached to adetection molecule, to possess (e.g., multiple) stabilizingmodifications, etc.

In one aspect, the invention provides an isolated double strandednucleic acid (dsNA) having a first and a second oligonucleotide strand,where the first strand is 27 to 49 nucleotide residues in length, andstarting from the first nucleotide (position 1) at the 5′ terminus ofthe first strand, positions 1 to 23 of the first strand areribonucleotides; where the second strand is 27 to 53 nucleotide residuesin length and includes 23 consecutive ribonucleotides that base pairwith the ribonucleotides of positions 1 to 23 of the first strand toform a duplex; the 5′ terminus of the first strand and the 3′ terminusof the second strand form a blunt end or a 1-4 nucleotide 3′ overhang;the 3′ terminus of the first strand and the 5′ terminus of the secondstrand form a blunt end; at least one of positions 24 to the 3′ terminalnucleotide residue of the first strand is a deoxyribonucleotide thatbase pairs with a deoxyribonucleotide of the second strand; and thesecond strand is sufficiently complementary to a target RNA along atleast 19 ribonucleotides of the second strand length to reduce targetgene expression when the dsNA is introduced into a mammalian cell.

In one embodiment, two or more nucleotide residues of positions 24 tothe 3′ terminal nucleotide residue of the first strand aredeoxyribonucleotides that base pair with deoxyribonucleotides of thesecond strand. In another embodiment, four or more nucleotide residuesof positions 24 to the 3′ terminal nucleotide residue of the firststrand are deoxyribonucleotides that base pair with deoxyribonucleotidesof the second strand. Optionally, six or more nucleotide residues, eightor more nucleotide residues, ten or more nucleotide residues, twelve ormore nucleotide residues, fourteen or more nucleotide residues, sixteenor more nucleotide residues, eighteen or more nucleotide residues, ortwenty or more nucleotide residues of positions 24 to the 3′ terminalnucleotide residue of the first strand are deoxyribonucleotides thatbase pair with deoxyribonucleotides of the second strand. In oneembodiment, the deoxyribonucleotides of the first strand that base pairwith the deoxyribonucleotides of the second strand are consecutivedeoxyribonucleotides. In another embodiment, two or more consecutivenucleotide residues of positions 24 to 27 of the first strand aredeoxyribonucleotides that base pair with deoxyribonucleotides of thesecond strand. Optionally, each of positions 24 and 25 of the firststrand is a deoxyribonucleotide that base pairs with adeoxyribonucleotide of the second strand. In a related embodiment, eachnucleotide residue of positions 24 to 27 of the first oligonucleotidestrand is a deoxyribonucleotide that base pairs with adeoxyribonucleotide of the second strand.

In one embodiment, the first strand is 29 to 49 nucleotides in length.In another embodiment, each nucleotide residue of positions 24 to 29 ofthe first oligonucleotide strand is a deoxyribonucleotide that basepairs with a deoxyribonucleotide of the second strand. In a furtherembodiment, the first strand is 31 to 49 nucleotides in length.Optionally, each nucleotide residue of positions 24 to 31 of the firstoligonucleotide strand is a deoxyribonucleotide that base pairs with adeoxyribonucleotide of the second strand. In an additional embodiment,the first strand is 33 to 49 nucleotides in length. Optionally, eachnucleotide residue of positions 24 to 33 of the first oligonucleotidestrand is a deoxyribonucleotide that base pairs with adeoxyribonucleotide of the second strand. In another embodiment, thefirst strand is 35 to 49 nucleotides in length. Optionally, eachnucleotide residue of positions 24 to 35 of the first oligonucleotidestrand is a deoxyribonucleotide that base pairs with adeoxyribonucleotide of the second strand. In another embodiment, thefirst strand is 37 to 49 nucleotides in length. Optionally, eachnucleotide residue of positions 24 to 37 of the first oligonucleotidestrand is a deoxyribonucleotide that base pairs with adeoxyribonucleotide of the second strand.

In an additional embodiment, positions 24 to the 3′ terminal nucleotideresidue of the first strand include between one and 25deoxyribonucleotide residues, and each of the deoxyribonucleotideresidues of the first strand base pairs with a deoxyribonucleotide ofthe second strand.

In one embodiment, the deoxyribonucleotides of the second strand thatbase pair with the deoxyribonucleotides of the first strand are notcomplementary to the target RNA.

In another embodiment, the second strand possesses a 3′ overhang of 1-4nucleotides in length. Optionally, the 3′ overhang is 1-3 nucleotides inlength, or, as a further option, 1-2 nucleotides in length. In oneembodiment, the nucleotides of the 3′ overhang include a modifiednucleotide. Optionally, the modified nucleotide residue is 2′-O-methyl,2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge,4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino or 2′-O-(N-methlycarbamate). Inone embodiment, the modified nucleotide of the 3′ overhang is a2′-O-methyl ribonucleotide. In a further embodiment, all nucleotides ofthe 3′ overhang are modified nucleotides. In a further embodiment, the3′ overhang is two nucleotides in length and the modified nucleotide ofthe 3′ overhang is a 2′-O-methyl modified ribonucleotide. In anotherembodiment, the second strand, starting from the nucleotide residue ofthe second strand that is complementary to the 5′ terminal nucleotideresidue of the first oligonucleotide strand, possesses unmodifiednucleotide residues at all positions from position 20 to the 5′ terminalresidue of the second strand.

In another embodiment, one or both of the first and second strandsincludes a 5′ phosphate.

In one embodiment, starting from the first nucleotide (position 1*) atthe 3′ terminus of the first strand, position 1*, 2* and/or 3* is adeoxyribonucleotide. In another embodiment, the first strand has adeoxyribonucleotide at position 1* from the 3′ terminus of the firststrand. In a related embodiment, the first strand hasdeoxyribonucleotides at positions 1* and 2* from the 3′ terminus of thefirst strand.

In another embodiment, the ultimate and penultimate residues of the 3′terminus of the first strand are deoxyribonucleotides and the ultimateand penultimate residues of the 5′ terminus of the second strand areribonucleotides.

In one embodiment, a nucleotide of the second or first oligonucleotidestrand is substituted with a modified nucleotide that directs theorientation of Dicer cleavage. In an additional embodiment, startingfrom the first nucleotide (position 1) at the 3′ terminus of the secondstrand, positions 1, 2, and 3 from the 3′ terminus of the second strandare modified nucleotides.

In one embodiment, the first strand has a nucleotide sequence that is atleast 80%, 90%, 95% or 100% complementary to the second strandnucleotide sequence.

In another embodiment, the 3′ terminal nucleotide residue of the firststrand is attached to the 5′ terminal nucleotide residue of the secondstrand by a nucleotide sequence. In one embodiment, the nucleotidesequence that attaches the 3′ terminal nucleotide residue of the firststrand and the 5′ terminal nucleotide residue of the second strandincludes a tetraloop. In another embodiment, the nucleotide sequencethat attaches the 3′ terminal nucleotide residue of the first strand andthe 5′ terminal nucleotide residue of the second strand includes ahairpin.

In a further embodiment, the first and second strands are joined by achemical linker. In a related embodiment, the 3′ terminus of the firststrand and the 5′ terminus of the second strand are joined by a chemicallinker.

In one embodiment, the dsNA is cleaved endogenously in a mammalian cellby Dicer. In another embodiment, the dsNA is cleaved endogenously in amammalian cell to produce a double-stranded nucleic acid of 19-23nucleotides in length that reduces target gene expression.

In an additional embodiment, the dsNA has a phosphate backbonemodification that is a phosphonate, a phosphorothioate or aphosphotriester.

In a further embodiment, the dsNA reduces target gene expression in amammalian cell in vitro by at least 10%, at least 50% or at least80-90%.

In one embodiment, the dsNA, when introduced into a mammalian cell,reduces target gene expression in comparison to a reference dsRNA thatdoes not possess a deoxyribonucleotide-deoxyribonucleotide base pair.

In an additional embodiment, the dsNA, when introduced into a mammaliancell, reduces target gene expression by at least 70% when transfectedinto the cell at a concentration of 1 nM or less, 200 pM or less, 100 pMor less, 50 pM or less, 20 pM or less, or 10 pM or less.

In another embodiment, at least 50% of the ribonucleotide residues ofthe dsNA are unmodified ribonucleotides. In an additional embodiment, atleast 50% of the ribonucleotide residues of the second strand areunmodified ribonucleotides.

In one embodiment, at least one of positions 24 to the 3′ terminalnucleotide residue of the first strand that is a deoxyribonucleotidethat base pairs with a deoxyribonucleotide of the second strand is anunmodified deoxyribonucleotide. In a related embodiment, both the atleast one of positions 24 to the 3′ terminal nucleotide residue of thefirst strand that is a deoxyribonucleotide that base pairs with adeoxyribonucleotide of the second strand and the deoxyribonucleotide ofthe second strand are unmodified deoxyribonucleotides. In anotherembodiment, at least 50% of all deoxyribonucleotides of the dsNA areunmodified deoxyribonucleotides.

In one embodiment, the second oligonucleotide strand, starting from thenucleotide residue of the second strand that is complementary to the 5′terminal nucleotide residue of the first oligonucleotide strand,includes alternating modified and unmodified nucleotide residues.

In certain embodiments, the target RNA is KRAS.

In another aspect, the invention provides an isolated double strandednucleic acid (dsNA) having a first and a second oligonucleotide strand,where the first strand: is 27 nucleotide residues in length and thesecond strand is 27-31 nucleotide residues in length; starting from thefirst nucleotide (position 1) at the 5′ terminus of the first strand,positions 1 to 23 of the first strand are ribonucleotides that base pairwith ribonucleotides of the second strand to form a duplex; each ofpositions 24 to 27 of the first strand is a deoxyribonucleotide thatbase pairs with a deoxyribonucleotide of the second strand; the 3′terminus of the first strand and the 5′ terminus of the second strandform a blunt end; and the second strand is sufficiently complementary toa target RNA along at least 19 ribonucleotides of the second strandlength to reduce target gene expression when the double stranded nucleicacid is introduced into a mammalian cell.

In an additional aspect, the invention provides an isolated doublestranded nucleic acid (dsNA) having a first oligonucleotide strand and asecond oligonucleotide strand, where the first strand is 29 nucleotideresidues in length and the second strand is 29-33 nucleotide residues inlength, where starting from the first nucleotide (position 1) at the 5′terminus of the first strand, positions 1 to 23 of the first strand areribonucleotides that base pair with ribonucleotides of the second strandto form a duplex; each of positions 24 to 29 of the first strand is adeoxyribonucleotide that base pairs with a deoxyribonucleotide of thesecond strand; the 3′ terminus of the first strand and the 5′ terminusof the second strand form a blunt end; and the second strand issufficiently complementary to a target RNA along at least 19ribonucleotides of the second strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell.

In another aspect, the invention provides an isolated double strandednucleic acid (dsNA) having a first oligonucleotide strand and a secondoligonucleotide strand, where the first strand is 31 nucleotide residuesin length and the second strand is 31-35 nucleotide residues in length,and starting from the first nucleotide (position 1) at the 5′ terminusof the first strand, positions 1 to 23 of the first strand areribonucleotides that base pair with ribonucleotides of the second strandto form a duplex; each of positions 24 to 31 of the first strand is adeoxyribonucleotide that base pairs with a deoxyribonucleotide of thesecond strand to form a duplex; the 3′ terminus of the first strand andthe 5′ terminus of the second strand form a blunt end; and the secondstrand is sufficiently complementary to a target RNA along at least 19ribonucleotides of the second strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell.

In one aspect, the invention provides an isolated double strandednucleic acid (dsNA) having a first oligonucleotide strand and a secondoligonucleotide strand, where the first strand is 33 nucleotide residuesin length and the second strand is 33-37 nucleotide residues in length,where starting from the first nucleotide (position 1) at the 5′ terminusof the first strand, positions 1 to 23 of the first strand areribonucleotides that base pair with ribonucleotides of the second strandto form a duplex; each of positions 24 to 33 of the first strand is adeoxyribonucleotide that base pairs with a deoxyribonucleotide of thesecond strand to form a duplex; the 3′ terminus of the first strand andthe 5′ terminus of the second strand form a blunt end; and the secondstrand is sufficiently complementary to a target RNA along at least 19ribonucleotides of the second strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell.

In another aspect, the invention provides an isolated double strandednucleic acid (dsNA) having a first oligonucleotide strand and a secondoligonucleotide strand, where the first strand is 35 nucleotide residuesin length and the second strand is 35-39 nucleotide residues in length,where starting from the first nucleotide (position 1) at the 5′ terminusof the first strand, positions 1 to 23 of the first strand areribonucleotides that base pair with ribonucleotides of the second strandto form a duplex; each of positions 24 to 35 of the first strand is adeoxyribonucleotide that base pairs with a deoxyribonucleotide of thesecond strand to form a duplex; the 3′ terminus of the first strand andthe 5′ terminus of the second strand form a blunt end; and the secondstrand is sufficiently complementary to a target RNA along at least 19ribonucleotides of the second strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell.

In a further aspect, the invention provides an isolated double strandednucleic acid (dsNA) having a first oligonucleotide strand and a secondoligonucleotide strand, where the first strand is 37 nucleotide residuesin length and the second strand is 37-41 nucleotide residues in length,where starting from the first nucleotide (position 1) at the 5′ terminusof the first strand, positions 1 to 23 of the first strand areribonucleotides that base pair with ribonucleotides of the second strandto form a duplex; each of positions 24 to 37 of the first strand is adeoxyribonucleotide that base pairs with a deoxyribonucleotide of thesecond strand to form a duplex; the 3′ terminus of the first strand andthe 5′ terminus of the second strand form a blunt end; and the secondstrand is sufficiently complementary to a target RNA along at least 19ribonucleotides of the second strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell.

In one embodiment, the deoxyribonucleotides of the second strand thatbase pair with the deoxyribonucleotides of the first strand are notcomplementary to the target RNA.

In one aspect, the invention provides an isolated double strandednucleic acid (dsNA) having a first and a second oligonucleotide strandwhere the first strand: is 27 nucleotide residues in length, andstarting from the first nucleotide (position 1) at the 5′ terminus ofthe first strand, positions 1 to 23 of the first strand areribonucleotides and positions 24 to 27 are deoxyribonucleotides; thesecond strand: is 29 nucleotide residues in length, starting from thefirst nucleotide (position 1) at the 5′ terminus of the second strand,positions 1 to 4 of the second strand are deoxyribonucleotides, and thesecond strand includes 23 consecutive ribonucleotides that base pairwith the ribonucleotides of positions 1 to 23 of the first strand toform a duplex; the 5′ terminus of the first strand and the 3′ terminusof the second strand form a 2 nucleotide 3′ overhang structure; the 3′terminus of the first strand and the 5′ terminus of the second strandform a blunt end; and the second strand is sufficiently complementary toa target RNA along at least 19 ribonucleotides of the second strandlength to reduce target gene expression when the double stranded nucleicacid is introduced into a mammalian cell.

In one embodiment, the nucleotides of the 3′ overhang are modifiednucleotides. In another embodiment, starting from the first nucleotide(position 1) at the 5′ terminus of the second strand, at least one ofpositions 13-27 is a modified ribonucleotide. In a further embodiment,starting from the first nucleotide (position 1) at the 5′ terminus ofthe second strand, positions 13, 15, 17, 19, 21, 23, 25 and 27 aremodified ribonucleotides.

In another aspect, the invention provides an isolated double strandednucleic acid (dsNA) having a first and a second oligonucleotide strand,where the first strand is 27 to 49 nucleotide residues in length, andstarting from the first nucleotide (position 1) at the 5′ terminus ofthe first strand, positions 1 to 23 of the first strand areribonucleotides; the second strand is 27 to 53 nucleotide residues inlength and includes 23 consecutive ribonucleotides that base pair withthe ribonucleotides of positions 1 to 23 of the first strand to form aduplex; the 5′ terminus of the first strand and the 3′ terminus of thesecond strand form a blunt end or 1-4 nucleotide overhang structure, the3′ terminus of the first strand and the 5′ terminus of the second strandform a blunt end, and at least one of positions 24 to the 3′ terminalnucleotide residue of the first strand is a phosphorothioate-modifiednucleotide (PS-NA) that base pairs with a deoxyribonucleotide of thesecond strand, with the second strand being sufficiently complementaryto a target RNA along at least 19 ribonucleotides of the second strandlength to reduce target gene expression when the double stranded nucleicacid is introduced into a mammalian cell.

In one embodiment, two or more nucleotide residues of positions 24 tothe 3′ terminal nucleotide residue of the first strand are PS-NAresidues that base pair with deoxyribonucleotides of the second strand.Optionally, four or more, six or more, eight or more, ten or more,twelve or more, or fifteen or more nucleotide residues of positions 24to the 3′ terminal nucleotide residue of the first strand are PS-NAresidues that base pair with deoxyribonucleotides of the second strand.In another embodiment, the deoxyribonucleotide of the second strand thatbase pairs with the PS-NA of the first strand is also a PS-NA.

In another aspect, the invention provides an isolated double strandednucleic acid (dsNA) having a first strand and a second strand where thefirst strand is 27 to 49 nucleotide residues in length and starting fromthe first nucleotide (position 1) at the 5′ terminus of the firststrand, positions 1 to 23 of the first strand are ribonucleotides; thesecond strand is 27 to 53 nucleotide residues in length and includes 23consecutive ribonucleotides that base pair with the ribonucleotides ofpositions 1 to 23 of the first strand to form a duplex; the 5′ terminusof the first strand and the 3′ terminus of the second strand form astructure that is either a blunt end or a 1-4 nucleotide 3′ overhang;the 3′ terminus of the first strand and the 5′ terminus of the secondstrand form a blunt end; at least one nucleotide of the second strandbase pairs with a deoxyribonucleotide of positions 24 to the 3′ terminalnucleotide residue of the first strand and is aphosphorothioate-modified nucleotide (PS-NA); and the second strand issufficiently complementary to a target RNA along at least 19ribonucleotides of the second strand length to reduce target geneexpression when the double stranded nucleic acid is introduced into amammalian cell.

In one embodiment, two or more nucleotide residues of the second strandbase pair with deoxyribonucleotides of positions 24 to the 3′ terminalnucleotide residue of the first strand and are PS-NA residues.Optionally, four or more, six or more, eight or more, ten or more,twelve or more, or fifteen or more nucleotide residues of the secondstrand base pair with deoxyribonucleotides of positions 24 to the 3′terminal nucleotide residue of the first strand and are PS-NA residues.In another embodiment, the dsNA includes two or more, three or more,four or more, five or more, six or more, seven or more, eight or more,nine or more, ten or more, eleven or more, twelve or more, thirteen ormore, fourteen or more, or fifteen or more total PS-NA residues.

In an additional aspect, the invention provides an isolated doublestranded nucleic acid (dsNA) having a first and a second oligonucleotidestrand, where the first strand is 27 to 49 nucleotide residues inlength, and starting from the first nucleotide (position 1) at the 5′terminus of the first strand, positions 1 to 23 of the first strand areribonucleotides; the second strand is 27 to 53 nucleotide residues inlength and includes 23 consecutive ribonucleotides that base pair withthe ribonucleotides of positions 1 to 23 of the first strand to form aduplex; the 5′ terminus of the first strand and the 3′ terminus of thesecond strand form a blunt end or 1-4 nucleotide overhang structure; the3′ terminus of the first strand and the 5′ terminus of the second strandform a blunt end; and at least one of positions 24 to the 3′ terminalnucleotide residue of the first strand is a deoxyribonucleotide thatbase pairs with a phosphorothioate-modified nucleotide (PS-NA) of thesecond strand, with the second strand being sufficiently complementaryto a target RNA along at least 19 ribonucleotides of the second strandlength to reduce target gene expression when the double stranded nucleicacid is introduced into a mammalian cell.

In one embodiment, two or more nucleotide residues of positions 24 tothe 3′ terminal nucleotide residue of the first strand aredeoxyribonucleotides that base pair with PS-NA residues of the secondstrand. Optionally, four or more, six or more, eight or more, ten ormore, twelve or more, or fifteen or more nucleotide residues ofpositions 24 to the 3′ terminal nucleotide residue of the first strandare deoxyribonucleotides that base pair with PS-NA residues of thesecond strand. In a further embodiment, the deoxyribonucleotide of thefirst strand that base pairs with the PS-NA of the second strand is alsoa PS-NA.

In another embodiment, the invention provides a method for reducingexpression of a target gene in a cell involving contacting a cell withan isolated dsNA as described in an amount effective to reduceexpression of a target gene in a cell in comparison to a referencedsRNA.

In an additional embodiment, the invention provides a method forreducing expression of a target gene in an animal that includes treatingan animal with an isolated dsNA as described in an amount effective toreduce expression of a target gene in a cell of the animal in comparisonto a reference dsRNA.

In one embodiment, the dsNA possesses enhanced pharmacokinetics whencompared to an appropriate control DsiRNA. In another embodiment, thedsNA possesses enhanced pharmacodynamics when compared to an appropriatecontrol DsiRNA. In an additional embodiment, the dsNA possesses reducedtoxicity when compared to an appropriate control DsiRNA. In a furtherembodiment, the dsNA possesses enhanced intracellular uptake whencompared to an appropriate control DsiRNA.

In a further embodiment, the invention provides a pharmaceuticalcomposition that includes an isolated dsNA as described in an amounteffective to reduce expression of a target gene in a cell in comparisonto a reference dsRNA and a pharmaceutically acceptable carrier, forreducing expression of a target gene in a cell of a subject. In anotherembodiment, the invention provides a method of synthesizing dsNA asdescribed, involving chemically or enzymatically synthesizing the dsNA.In an additional embodiment, the invention provides a kit that includesa dsNA as described, and instructions for its use.

In one aspect, the invention provides an isolated dsNA as shown in FIG.30.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic representation of the processing of a Dicersubstrate inhibitory RNA agent (“DsiRNA”). The protein Dicer isrepresented by the large rectangle, with the PAZ (Piwi/Argonaute/Zwille)domain of Dicer also indicated. The PAZ domain binds the two-baseoverhang and the 3′-OH (hydroxyl group) at the 3′ end of the guide(antisense) strand, and each strand of the dsRNA duplex is cleaved byseparate RNase III domains (black triangles). Substitution of 2 bases ofDNA for RNA at the 3′ end of the passenger (sense) strand forms atwo-base long RNA/DNA duplex blunt end, which reduces or eliminatesbinding affinity for PAZ. Cleavage of the DsiRNA typically yields a19mer duplex with 2-base overhangs at each end. FIG. 1B shows that theaddition of four bases of DNA duplex to the DsiRNA had no apparentinhibitory effect upon Dicer cleavage. The bases inserted into thisexample of an anti-HPRT DsiRNA (heavy black bars and arrows) were notcomplementary to the HPRT target sequence.

FIG. 2A presents histogram data showing the robust efficacy of DsiRNAagents possessing base paired deoxyribonucleotides in a duplexed regionlocated 3′ of the Dicer cleavage site of the sense strand/5′ of theDicer cleavage site of the antisense strand (“Right-extended DsiRNAagents”). DsiRNA duplexes were transfected into HeLa cells at a fixedconcentration of 20 nM, and HPRT expression levels were measured 24hours later. Transfections were performed in duplicate, and eachduplicate was assayed in triplicate for HPRT expression by qPCR. Errorbars are the standard error. Duplex 1 targeted HPRT and was a 25/27merconfiguration overhanging RNA/blunt two-DNA substitution as described inRose et al. NAR 2005. All other duplexes were longer than Duplex 1 dueto the insertion of bases that were not complementary to the HPRT targetregion. The length of the inserted sequence ranged from two bases(Duplex 2) to eight bases (Duplexes 6, 7, and 8). FIG. 2B shows duplexnumbers, sequences and chemical modification patterns for agents forwhich data is presented in FIG. 2A. UPPER case=unmodified RNA, Bold,underlined=2′-O-methyl RNA, lower case=DNA, bold lowercase=phosphorothioate-modified DNA (PS-DNA). A general description ofeach duplex and the overall configuration is shown at right.

FIGS. 3A and 3B show that DNA-extended DsiRNA agents were more effectivethan corresponding RNA-extended DsiRNA agents at low concentrations. Anoptimized 27/29mer DsiRNA duplex targeting HPRT was compared to amodified duplex in a dose-response series at 10.0 nanomolar (nM), 1.0nanomolar (nM) and 100 picomolar (100 pM or 0.1 nM), with efficacy ofknockdown of HPRT mRNA levels assessed in HeLa cells. Duplexconcentrations shown represent the final concentration ofoligonucleotides in the transfection mixture and culture medium asdescribed in the Examples. Duplex identities are indicated below thebars (1, 2, 3), with the “C” bar representing baseline HPRT expressionin untreated cells. FIG. 3B shows the sequences and chemicalmodification patterns of those duplexes depicted in FIG. 3A. UPPERcase=unmodified RNA, Bold, underlined=2′-O-methyl RNA, and lowercase=DNA. DsiRNA 1 was a derivative of a previously reported active25/27mer DsiRNA duplex (HPRT-1, Rose et al. NAR 2005, Collingwood et al.2008, see also FIG. 2A above), but contained an insertion of two basesin each strand, which extended the oligonucleotide duplex to a 27/29mer(heavy black bars denote inserted base pairs). Duplex 2 was identical insequence to duplex 1, but the two base pair insertion (heavy blackbars), including two additional nucleosides of both passenger strand(sense sequence) and guide strand (antisense sequence) were synthesizedas DNA. Thus, duplex 2 terminated in 4 DNA by (base pairs) at the 5′ endof the guide strand, in contrast to previously reported two base DNAsubstitutions at the 3′ end of the passenger (sense) strand (Rose et al,2005). Duplex 3 (mismatch (MM) control) was derived from the optimizedHPRT-1 duplex, but synthesized with mismatches indicated by arrows. Thebase composition and chemical modification of each strand and the basesequences and overhang or blunt structure at the ends of duplex 3 wereheld constant relative to the optimized HPRT-1 duplex in order tocontrol for non-targeted chemical effects (see FIG. 5 below).

FIGS. 4A-4D show that modified DsiRNA duplexes extended by two to eightbase paired deoxyribonucleosides were more effective at reducing HPRTtarget mRNA levels than corresponding ribonucleoside-extended DsiRNAagents. FIG. 4A shows HPRT target gene mRNA levels for cells treatedwith 1 nM modified DsiRNA agents. FIG. 4B shows HPRT target gene mRNAlevels for cells treated with 100 pM modified DsiRNA agents. FIG. 4Cshows HPRT target gene mRNA levels for cells treated with 10 pM modifiedDsiRNA agents. FIG. 4D shows the sequences and chemical modificationpatterns of those duplexes depicted in FIGS. 4A-4C. Inserted sequences(heavy bars beneath the duplexes) did not match the HPRT mRNA targetregion. UPPER case=unmodified RNA, Bold, underlined=2′-O-methyl RNA,lower case=DNA. U=untreated cells.

FIG. 5A shows HPRT target mRNA inhibition results for a series ofmodified duplexes of increasing length administered at a fixedconcentration of 100 pM. FIG. 5B shows duplex numbers, sequences andchemical modification patterns for agents for which data is presented inFIG. 5A. Duplex 1 was an optimized 25/27mer DsiRNA containing chemicalmodifications, a two-base overhang at the 3′-end of the guide(antisense) strand and two DNA substitutions and a blunt end at the3′-end of the passenger (sense) strand (Collingwood et al. 2008). Basesnon-complementary to HPRT mRNA were inserted two bases at a time aseither RNA (duplexes 2 through 5) or DNA (duplexes 6 through 9),increasing total duplex configurations from 27/29mers to 33/35mers.UPPER case=unmodified RNA, Bold, underlined=2′-O-methyl RNA, lowercase=DNA. U=untreated cells. UPPER case=unmodified RNA, Bold,underlined=2′-O-methyl RNA, lower case=DNA.

FIG. 6 shows the structure and predicted Dicer-mediated processing of a“25/27mer DsiRNA” agent (top) and an exemplary “Left-extended” DsiRNAagent (bottom) which contains a mismatch residue (G:U) within the dsRNAduplex sequence. UPPER case=RNA residues; lower case=DNA residues.

FIG. 7 shows the structures of a series of DNA-extended duplexes, withpictured duplexes alternately right- or left-extended with 5 base pairDNA sequences. Mismatches are introduced within both forms of extendedDsiRNA agents as indicated, with numbering of such mismatches proceedingin the 3′ direction from position 1 of the second strand, which is thepredicted 5′ terminal RNA residue of the second strand after Dicercleavage.

FIG. 8 depicts the results of an initial round of testing of theinhibitory activity of right- and left-extended agents shown in FIG. 7.For comparisons between right- versus left-extended parent molecules,right- versus left-extended agents harboring a mismatch at position 14,right-versus left-extended agents possessing a mismatch at position 16,and right- versus left-extended agents harboring a mismatch at bothpositions 14 and 18, left-extended agents were surprisingly observed tobe more effective at gene silencing than corresponding right-extendedagents. (100 pM of each indicated duplex was transfected into HeLa cellsfor all such experiments and % of KRAS target mRNA remaining wasassessed at 24 hours.)

FIG. 9 depicts the result of a second round of experiments performedwith the agents shown in FIG. 7, showing that left-extended agents werereproducibly more effective target mRNA silencing agents thanright-extended agents in three of the four instances which wereinitially observed to show such a bias in favor of left-extended agents.Inhibitory results for a non-extended 25/27mer DsiRNA are also shown(“Opt” 25/27mer).

FIG. 10 shows the structure of a series of DsiRNA agents designed tosilence an HPRT target mRNA, and inhibitory efficacies of such agents incell culture. Capital letters indicate ribonucleotides; lower caseletters indicate deoxyribonucleotides; bolded lower case lettersindicate phosphorothioates (PS-NAs); bolded and underlined uppercaseletters indicate 2′-O-methyl modified nucleotides; the bolded uppercaseletter of agent DP1065P/DP1067G indicates the site of a mismatchednucleotide (with respect to the sense strand) within the “seed” regionsequence of the antisense strand of the DsiRNA agent.

FIG. 11 shows that phosphorothioate modified “right-extended” DsiRNAsretain target HPRT1 gene inhibitory efficacy, and also indicates thatpassenger strand extended residues might tolerate phosphorothioatemodification better than guide strand extended residues while retainingtarget gene inhibitory activity. In vitro Dicer cleavage assays (leftlane=untreated; right lane=Dicer enzyme treated) are also shown for allextended DsiRNAs. Capital letters indicate ribonucleotides; lower caseletters indicate deoxyribonucleotides, while bolded lower case lettersindicate phosphorothioate-modified deoxyribonucleotides.

FIG. 12 depicts the structures of control and “right-extended” DsiRNAsof the invention targeting the “KRAS-200” site within the KRAStranscript. Capital letters indicate ribonucleotides; lower case lettersindicate deoxyribonucleotides.

FIG. 13 shows the KRAS inhibitory efficacies observed for the DsiRNAstructures of FIG. 12 in vitro.

FIG. 14 depicts the structures of control and “right-extended” DsiRNAsof the invention targeting the “KRAS-909” site within the KRAStranscript. Capital letters indicate ribonucleotides; lower case lettersindicate deoxyribonucleotides.

FIG. 15 shows the KRAS inhibitory efficacies observed for the DsiRNAstructures of FIG. 14 in vitro.

FIG. 16 depicts the structures of control and “right-extended” DsiRNAsof the invention targeting the “KRAS-249” site within the KRAStranscript, including modification patterns of such DsiRNAs. Capitalletters indicate ribonucleotides; lower case letters indicatedeoxyribonucleotides, while bolded lower case letters indicatephosphorothioate-modified deoxyribonucleotides. Underlined capitalletters indicate 2′-O-methyl-modified ribonucleotides.

FIG. 17 depicts the structures of control and “right-extended” DsiRNAsof the invention targeting the “KRAS-516” site within the KRAStranscript, including modification patterns of such DsiRNAs. Capitalletters indicate ribonucleotides; lower case letters indicatedeoxyribonucleotides, while bolded lower case letters indicatephosphorothioate-modified deoxyribonucleotides. Underlined capitalletters indicate 2′-O-methyl-modified ribonucleotides.

FIG. 18 depicts the structures of control and “right-extended” DsiRNAsof the invention targeting the “KRAS-909” site within the KRAStranscript, including modification patterns of such DsiRNAs. Capitalletters indicate ribonucleotides; lower case letters indicatedeoxyribonucleotides, while bolded lower case letters indicatephosphorothioate-modified deoxyribonucleotides. Underlined capitalletters indicate 2′-O-methyl-modified ribonucleotides.

FIG. 19 shows in vitro KRAS inhibitory efficacy results obtained for the“right-extended” DsiRNAs of FIGS. 16-18. Results were obtained in HeLacells contacted with the indicated DsiRNAs at 0.1 nM concentration,assayed at 24 hours post-RNAiMAX™ treatment. Capital letters indicateribonucleotides; lower case letters indicate deoxyribonucleotides, whilebolded lower case letters indicate phosphorothioate-modifieddeoxyribonucleotides.

FIG. 20 depicts the structures of 25/27mer “KRAS-249” site targetingDsiRNAs which were assessed for mismatch residue tolerance. Closedarrows indicate projected Dicer enzyme cleavage sites, while open arrowindicates projected Ago2 cleavage site within target strand sequencecorresponding to passenger strand DsiRNA sequence shown. Capital lettersindicate ribonucleotides; lower case letters indicatedeoxyribonucleotides. Bolded capital letters indicate sites oftarget-mismatched residues of guide strand (and complementary residuesof passenger strand, where applicable), with such target-mismatchedresidues obtained by “flipping” individual residues between guide andpassenger strand during DsiRNA design. Horizontal bracket withinDP1301P/DP1302G duplex indicates “seed region” of this duplex (with seedregions of all other DsiRNA structures occurring in the samevertically-aligned position).

FIG. 21 shows in vitro KRAS inhibitory efficacy results obtained for theDsiRNAs of FIG. 20. Results were obtained in HeLa cells contacted withthe indicated DsiRNAs at 0.1 nM concentration, assayed at 24 hourspost-RNAiMAX™ treatment.

FIG. 22 shows single dose (10 mg/kg) in vivo KRAS inhibitory efficacyresults in liver tissue for an unmodified 25/27mer “KRAS-249” sitetargeting DsiRNA (“K249”), a 2′-O-methyl-modified form of this 25/27mer(“KRAS-249M”) and a DNA-extended form of this modified DsiRNA(“K249DNA”, shown in FIG. 16 as “K249D”; “5% Glu”=5% glucose control).

FIG. 23 shows single dose (10 mg/kg) in vivo KRAS inhibitory efficacyresults in kidney tissue for an unmodified 25/27mer “KRAS-249” sitetargeting DsiRNA (“K249”), a 2′-O-methyl-modified form of this 25/27mer(“KRAS-249M”) and a DNA-extended form of this modified DsiRNA(“K249DNA”, shown in FIG. 16 as “K249D”; “5% Glu”=5% glucose control).

FIG. 24 shows single dose (10 mg/kg) in vivo KRAS inhibitory efficacyresults in spleen tissue for an unmodified 25/27mer “KRAS-249” sitetargeting DsiRNA (“K249”), a 2′-O-methyl-modified form of this 25/27mer(“KRAS-249M”) and a DNA-extended form of this modified DsiRNA(“K249DNA”, shown in FIG. 16 as “K249D”; “5% Glu”=5% glucose control).

FIG. 25 shows single dose (10 mg/kg) in vivo KRAS inhibitory efficacyresults in lymph node tissue for an unmodified 25/27mer “KRAS-249” sitetargeting DsiRNA (“K249”), a 2′-O-methyl-modified form of this 25/27mer(“KRAS-249M”) and a DNA-extended form of this modified DsiRNA(“K249DNA”, shown in FIG. 16 as “K249D”; “5% Glu”=5% glucose control).

FIG. 26 shows multi-dose (2 mg/kg, administered a total of four times,with each administration performed at three day intervals) in vivo KRASinhibitory efficacy results in liver tissue for a 2′-O-methyl-modifiedform of a 25/27mer “KRAS-249” site targeting DsiRNA (“KRAS-249M”) and aDNA-extended form of this modified DsiRNA (“K249D”, as shown in FIG.16).

FIG. 27 shows multi-dose (2 mg/kg, administered a total of four times,with each administration performed at three day intervals) in vivo KRASinhibitory efficacy results in lung tissue for a 2′-O-methyl-modifiedform of a 25/27mer “KRAS-249” site targeting DsiRNA (“KRAS-249M”) and aDNA-extended form of this modified DsiRNA (“K249D”, as shown in FIG.16).

FIG. 28 shows multi-dose (2 mg/kg, administered a total of four times,with each administration performed at three day intervals) in vivo KRASinhibitory efficacy results in spleen tissue for a 2′-O-methyl-modifiedform of a 25/27mer “KRAS-249” site targeting DsiRNA (“KRAS-249M”) and aDNA-extended form of this modified DsiRNA (“K249D”, as shown in FIG.16).

FIG. 29 shows multi-dose (2 mg/kg, administered a total of four times,with each administration performed at three day intervals) in vivo KRASinhibitory efficacy results in kidney tissue for a 2′-O-methyl-modifiedform of a 25/27mer “KRAS-249” site targeting DsiRNA (“KRAS-249M”) and aDNA-extended form of this modified DsiRNA (“K249D”, as shown in FIG.16).

FIG. 30 shows exemplary structures of “right extended” DsiRNA agentsthat form a blunt end between the 3′ terminus of the first strand and 5′terminus of the second strand. Upper case letters indicateribonucleotides; lower case characters denote deoxyribonucleotides; opentriangle denotes a site within the sequence of the first strand (here,the sense strand) corresponding to the Ago2 cleavage site within thetarget RNA; filled triangles indicate projected sites of Dicer cleavage;and [#] denotes a duplex region of four to sixteen or more base pairs inlength which comprises at least onedeoxyribonucleotide-deoxyribonucleotide base pair. (In alternativeembodiments, [#] indicates a duplex region of four to sixteen or morebase pairs in length which comprises at least four deoxyribonucleotidesbut is not required to possess a deoxyribonucleotide-deoxyribonucleotidebase pair.) Nucleotide position numbering is also shown.

FIG. 31 shows an exemplary structure of a “right extended” DsiRNA agentthat possesses a 3′-terminal overhang of the first strand relative tothe 5′ terminus of the second strand. Upper case letters indicateribonucleotides; lower case characters denote deoxyribonucleotides; opentriangle denotes a site within the sequence of the first strand (here,the sense strand) corresponding to the Ago2 cleavage site within thetarget RNA; filled triangles indicate projected sites of Dicer cleavage;and [#] denotes a duplex region of four to sixteen or more base pairs inlength which comprises at least onedeoxyribonucleotide-deoxyribonucleotide base pair. (In alternativeembodiments, [#] indicates a duplex region of four to sixteen or morebase pairs in length which comprises at least four deoxyribonucleotidesbut is not required to possess a deoxyribonucleotide-deoxyribonucleotidebase pair.) Nucleotide position numbering is also shown.

FIG. 32 shows an exemplary structure of a “right extended” DsiRNA agentthat forms a fray at the 3′-terminus of the first strand andcorresponding 5′ terminus of the second strand. Upper case lettersindicate ribonucleotides; lower case characters denotedeoxyribonucleotides; open triangle denotes a site within the sequenceof the first strand (here, the sense strand) corresponding to the Ago2cleavage site within the target RNA; filled triangles indicate projectedsites of Dicer cleavage; and [#] denotes a duplex region of four tosixteen or more base pairs in length which comprises at least onedeoxyribonucleotide-deoxyribonucleotide base pair. (In alternativeembodiments, [#] indicates a duplex region of four to sixteen or morebase pairs in length which comprises at least four deoxyribonucleotidesbut is not required to possess a deoxyribonucleotide-deoxyribonucleotidebase pair.) Nucleotide position numbering is also shown.

FIG. 33 shows exemplary structures of “right extended” DsiRNA agentsthat form a blunt end between the 3′ terminus of the first strand and 5′terminus of the second strand, and that also possess mismatched residueswithin antisense strand sequences which are projected to be retainedwithin the interference agent following Dicer cleavage. Upper caseletters indicate ribonucleotides; lower case characters denotedeoxyribonucleotides; open triangle denotes a site within the sequenceof the first strand (here, the sense strand) corresponding to the Ago2cleavage site within the target RNA; filled triangles indicate projectedsites of Dicer cleavage; and [#] denotes a duplex region of four tosixteen or more base pairs in length which comprises at least onedeoxyribonucleotide-deoxyribonucleotide base pair. (In alternativeembodiments, [#] indicates a duplex region of four to sixteen or morebase pairs in length which comprises at least four deoxyribonucleotidesbut is not required to possess a deoxyribonucleotide-deoxyribonucleotidebase pair.) Seed and mismatch regions of the antisense strand, as wellas nucleotide position numbering of each strand is also shown. Theunderlined antisense residue of the bottom agent indicates a nucleotidewhich base pairs with the sense strand of the DsiRNA agent, yet isprojected to form a mismatch with the target RNA.

FIG. 34 shows exemplary structures of “right extended” DsiRNA agentsthat possess a 3′-terminal overhang of the first strand relative to the5′ terminus of the second strand, and that also possess mismatchedresidues within antisense strand sequences which are projected to beretained within the interference agent following Dicer cleavage. Uppercase letters indicate ribonucleotides; lower case characters denotedeoxyribonucleotides; open triangle denotes a site within the sequenceof the first strand (here, the sense strand) corresponding to the Ago2cleavage site within the target RNA; filled triangles indicate projectedsites of Dicer cleavage; and [#] denotes a duplex region of four tosixteen or more base pairs in length which comprises at least onedeoxyribonucleotide-deoxyribonucleotide base pair. (In alternativeembodiments, [#] indicates a duplex region of four to sixteen or morebase pairs in length which comprises at least four deoxyribonucleotidesbut is not required to possess a deoxyribonucleotide-deoxyribonucleotidebase pair.) Seed and mismatch regions of the antisense strand, as wellas nucleotide position numbering of each strand is also shown. Theunderlined antisense residue of the bottom agent indicates a nucleotidewhich base pairs with the sense strand of the DsiRNA agent, yet isprojected to form a mismatch with the target RNA.

FIG. 35 shows exemplary structures of “right extended” DsiRNA agentsthat form a fray at the 3′-terminus of the first strand andcorresponding 5′ terminus of the second strand, and that also possessmismatched residues within antisense strand sequences which areprojected to be retained within the interference agent following Dicercleavage. Upper case letters indicate ribonucleotides; lower casecharacters denote deoxyribonucleotides; open triangle denotes a sitewithin the sequence of the first strand (here, the sense strand)corresponding to the Ago2 cleavage site within the target RNA; filledtriangles indicate projected sites of Dicer cleavage; and [#] denotes aduplex region of four to sixteen or more base pairs in length whichcomprises at least one deoxyribonucleotide-deoxyribonucleotide basepair. (In alternative embodiments, [#] indicates a duplex region of fourto sixteen or more base pairs in length which comprises at least fourdeoxyribonucleotides but is not required to possess adeoxyribonucleotide-deoxyribonucleotide base pair.) Seed and mismatchregions of the antisense strand, as well as nucleotide positionnumbering of each strand is also shown. The underlined antisense residueof the bottom agent indicates a nucleotide which base pairs with thesense strand of the DsiRNA agent, yet is projected to form a mismatchwith the target RNA.

FIG. 36 shows exemplary structures of “left extended” DsiRNA agents thatform a blunt end between the 3′ terminus of the first strand and 5′terminus of the second strand. Upper case letters indicateribonucleotides; lower case characters denote deoxyribonucleotides; opentriangle denotes a site within the sequence of the first strand (here,the sense strand) corresponding to the Ago2 cleavage site within thetarget RNA; filled triangles indicate projected sites of Dicer cleavage;and [#] denotes a duplex region of four to sixteen or more base pairs inlength which comprises at least onedeoxyribonucleotide-deoxyribonucleotide base pair. (In alternativeembodiments, [#] indicates a duplex region of four to sixteen or morebase pairs in length which comprises at least four deoxyribonucleotidesbut is not required to possess a deoxyribonucleotide-deoxyribonucleotidebase pair.) Nucleotide position numbering is also shown.

FIG. 37 shows exemplary structures of “left extended” DsiRNA agents thatpossess a 3′-terminal overhang of the first strand relative to the 5′terminus of the second strand. Upper case letters indicateribonucleotides; lower case characters denote deoxyribonucleotides; opentriangle denotes a site within the sequence of the first strand (here,the sense strand) corresponding to the Ago2 cleavage site within thetarget RNA; filled triangles indicate projected sites of Dicer cleavage;and [#] denotes a duplex region of four to sixteen or more base pairs inlength which comprises at least onedeoxyribonucleotide-deoxyribonucleotide base pair. (In alternativeembodiments, [#] indicates a duplex region of four to sixteen or morebase pairs in length which comprises at least four deoxyribonucleotidesbut is not required to possess a deoxyribonucleotide-deoxyribonucleotidebase pair.) Nucleotide position numbering is also shown.

FIG. 38 shows an exemplary structure of a “left extended” DsiRNA agentthat forms a fray at the 3′-terminus of the first strand andcorresponding 5′ terminus of the second strand. Upper case lettersindicate ribonucleotides; lower case characters denotedeoxyribonucleotides; open triangle denotes a site within the sequenceof the first strand (here, the sense strand) corresponding to the Ago2cleavage site within the target RNA; filled triangles indicate projectedsites of Dicer cleavage; and [#] denotes a duplex region of four tosixteen or more base pairs in length which comprises at least onedeoxyribonucleotide-deoxyribonucleotide base pair. (In alternativeembodiments, [#] indicates a duplex region of four to sixteen or morebase pairs in length which comprises at least four deoxyribonucleotidesbut is not required to possess a deoxyribonucleotide-deoxyribonucleotidebase pair.) Nucleotide position numbering is also shown.

FIG. 39 shows exemplary structures of “left extended” DsiRNA agents thatform a blunt end between the 3′ terminus of the first strand and 5′terminus of the second strand, and that also possess mismatched residueswithin antisense strand sequences which are projected to be retainedwithin the interference agent following Dicer cleavage. Upper caseletters indicate ribonucleotides; lower case characters denotedeoxyribonucleotides; open triangle denotes a site within the sequenceof the first strand (here, the sense strand) corresponding to the Ago2cleavage site within the target RNA; filled triangles indicate projectedsites of Dicer cleavage; and [#] denotes a duplex region of four tosixteen or more base pairs in length which comprises at least onedeoxyribonucleotide-deoxyribonucleotide base pair. (In alternativeembodiments, [#] indicates a duplex region of four to sixteen or morebase pairs in length which comprises at least four deoxyribonucleotidesbut is not required to possess a deoxyribonucleotide-deoxyribonucleotidebase pair.) Seed and mismatch regions of the antisense strand, as wellas nucleotide position numbering of each strand is also shown. Theunderlined antisense residue of the lower two agents indicates anucleotide which base pairs with the sense strand of the DsiRNA agent,yet is projected to form a mismatch with the target RNA.

FIG. 40 shows exemplary structures of “left extended” DsiRNA agents thatpossess a 3′-terminal overhang of the first strand relative to the 5′terminus of the second strand, and that also possess mismatched residueswithin antisense strand sequences which are projected to be retainedwithin the interference agent following Dicer cleavage. Upper caseletters indicate ribonucleotides; lower case characters denotedeoxyribonucleotides; open triangle denotes a site within the sequenceof the first strand (here, the sense strand) corresponding to the Ago2cleavage site within the target RNA; filled triangles indicate projectedsites of Dicer cleavage; and [#] denotes a duplex region of four tosixteen or more base pairs in length which comprises at least onedeoxyribonucleotide-deoxyribonucleotide base pair. (In alternativeembodiments, [#] indicates a duplex region of four to sixteen or morebase pairs in length which comprises at least four deoxyribonucleotidesbut is not required to possess a deoxyribonucleotide-deoxyribonucleotidebase pair.) Seed and mismatch regions of the antisense strand, as wellas nucleotide position numbering of each strand is also shown. Theunderlined antisense residue of the middle agent indicates a nucleotidewhich base pairs with the sense strand of the DsiRNA agent, yet isprojected to form a mismatch with the target RNA.

FIG. 41 shows exemplary structures of “left extended” DsiRNA agents thatform a fray at the 3′-terminus of the first strand and corresponding 5′terminus of the second strand, and that also possess mismatched residueswithin antisense strand sequences which are projected to be retainedwithin the interference agent following Dicer cleavage. Upper caseletters indicate ribonucleotides; lower case characters denotedeoxyribonucleotides; open triangle denotes a site within the sequenceof the first strand (here, the sense strand) corresponding to the Ago2cleavage site within the target RNA; filled triangles indicate projectedsites of Dicer cleavage; and [#] denotes a duplex region of four tosixteen or more base pairs in length which comprises at least onedeoxyribonucleotide-deoxyribonucleotide base pair. (In alternativeembodiments, [#] indicates a duplex region of four to sixteen or morebase pairs in length which comprises at least four deoxyribonucleotidesbut is not required to possess a deoxyribonucleotide-deoxyribonucleotidebase pair.) Seed and mismatch regions of the antisense strand, as wellas nucleotide position numbering of each strand is also shown. Theunderlined antisense residue of the bottom agent indicates a nucleotidewhich base pairs with the sense strand of the DsiRNA agent, yet isprojected to form a mismatch with the target RNA.

FIGS. 42A-42C show exemplary structures of “right extended” DsiRNAagents. Upper case letters indicate ribonucleotides; lower casecharacters denote deoxyribonucleotides; open triangle denotes a sitewithin the sequence of the top strand (here, the sense strand)corresponding to the Ago2 cleavage site within the target RNA; filledtriangles indicate projected sites of Dicer cleavage; and nucleotideposition numbering is also shown.

FIGS. 43A-43C show exemplary structures of “left extended” DsiRNAagents. Upper case letters indicate ribonucleotides; lower casecharacters denote deoxyribonucleotides; open triangle denotes a sitewithin the sequence of the top strand (here, the sense strand)corresponding to the Ago2 cleavage site within the target RNA; filledtriangles indicate projected sites of Dicer cleavage; and nucleotideposition numbering is also shown.

DETAILED DESCRIPTION

The invention provides compositions and methods for reducing expressionof a target gene in a cell, involving contacting a cell with an isolateddouble stranded nucleic acid (dsNA) in an amount effective to reduceexpression of a target gene in a cell. The dsNAs of the inventionpossess a pattern of deoxyribonucleotides (in most embodiments, thepattern comprises at least one deoxyribonucleotide-deoxyribonucleotidebase pair) designed to direct the site of Dicer enzyme cleavage withinthe dsNA molecule. The deoxyribonucleotide pattern of the dsNA moleculesof the invention is located within a region of the dsNA that can beexcised via Dicer cleavage to generate an active siRNA agent that nolonger contains the deoxyribonucleotide pattern (e.g., in mostembodiments, the deoxyribonucleotide pattern comprises one or moredeoxyribonucleotide-deoxyribonucleotide base pairs). Surprisingly, asdemonstrated herein, DNA:DNA-extended Dicer-substrate siRNAs (DsiRNAs)were more effective RNA inhibitory agents than corresponding RNA:DNA- orRNA:RNA-extended DsiRNAs.

It was also surprising to discover that DsiRNAs comprising DNA:DNAextensions which were positioned at the 5′ end of the first strand andcorresponding 3′ end of the second strand of a dsRNA DsiRNA agent (wherethe second strand is complementary to a sufficient region of target RNAsequence to serve as an effective guide strand sequence of an RNAi agent(antisense to the target RNA)) constituted effective—and in manyinstances enhanced—inhibitory agents.

The surprising discovery that DNA-extended DsiRNA agents do not exhibitdecreases in efficacy as duplex length increases allows for thegeneration of DsiRNAs that remain effective while providing greaterspacing for, e.g., attachment of DsiRNAs to additional and/or distinctfunctional groups, inclusion/patterning of stabilizing modifications(e.g., PS-NA moieties) or other forms of modifications capable of addingfurther functionality and/or enhancing, e.g., pharmacokinetics,pharmacodynamics or biodistribution of such agents, as compared to dsRNAagents of corresponding length that do not contain such double strandedDNA-extended domains.

The advantage provided by the newfound ability to lengthenDsiRNA-containing dsNA duplexes while retaining activity of apost-Dicer-processed siRNA agent at levels greater than dsRNA duplexesof similar length is emphasized by the results presented herein, whichshow that complete phosphorothioate (PS) modification of all nucleotidesof a double-stranded DNA:DNA region of an extended DsiRNA agentcompletely abolished silencing activity (see duplex #8 of FIGS. 2A and2B). The ability to extend DsiRNA agents without observing acorresponding reduction in RNA silencing activity can also allow forinclusion of, e.g., more modified nucleotides within a single moleculethat still retains RNA silencing activity than could otherwise beachieved were such modified nucleotides not allowed such spacing (inview of the inhibitory effect associated with certain modifications whenpresent in a tandem series—e.g., tandem PS or 2′-O-methylmodifications). Similarly, the ability to include longer duplexextensions in such DsiRNA-containing agents while retaining RNAinhibitory function can also allow for certain functional groups to beattached to such agents that would otherwise not be possible, because ofthe ability of such functional groups to interfere with RNA silencingactivity when present in tighter configurations.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

As used herein, the term “nucleic acid” refers to deoxyribonucleotides,ribonucleotides, or modified nucleotides, and polymers thereof insingle- or double-stranded form. The term encompasses nucleic acidscontaining known nucleotide analogs or modified backbone residues orlinkages, which are synthetic, naturally occurring, and non-naturallyoccurring, which have similar binding properties as the referencenucleic acid, and which are metabolized in a manner similar to thereference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs).

As used herein, “nucleotide” is used as recognized in the art to includethose with natural bases (standard), and modified bases well known inthe art. Such bases are generally located at the 1′ position of anucleotide sugar moiety. Nucleotides generally comprise a base, sugarand a phosphate group. The nucleotides can be unmodified or modified atthe sugar, phosphate and/or base moiety, (also referred tointerchangeably as nucleotide analogs, modified nucleotides, non-naturalnucleotides, non-standard nucleotides and other; see, e.g., Usman andMcSwiggen, supra; Eckstein, et al., International PCT Publication No. WO92/07065; Usman et al, International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach, et al, Nucleic Acids Res. 22:2183,1994. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, hypoxanthine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

As used herein, a “double-stranded nucleic acid” or “dsNA” is a moleculecomprising two oligonucleotide strands which form a duplex. A dsNA maycontain ribonucleotides, deoxyribonucleotides, modified nucleotides, andcombinations thereof. The double-stranded NAs of the instant inventionare substrates for proteins and protein complexes in the RNAinterference pathway, e.g., Dicer and RISC. An exemplary structure ofone form of dsNA of the invention is shown in FIG. 1A, and suchstructures characteristically comprise an RNA duplex in a region that iscapable of functioning as a Dicer substrate siRNA (DsiRNA) and a DNAduplex comprising at least one deoxyribonucleotide, which is located ata position 3′ of the projected Dicer cleavage site of the first strandof the DsiRNA/DNA agent, and is base paired with a cognatedeoxyribonucleotide of the second strand, which is located at a position5′ of the projected Dicer cleavage site of the second strand of theDsiRNA/DNA agent. In alternative embodiments, the instant inventionprovides a structure that characteristically comprises an RNA duplexwithin a region that is capable of functioning as a Dicer substratesiRNA (DsiRNA) and a DNA duplex comprising at least onedeoxyribonucleotide, which is located at a position 5′ of the projectedDicer cleavage site of the first strand of the DsiRNA/DNA agent, and isbase paired with a cognate deoxyribonucleotide of the second strand,which is located at a position 3′ of the projected Dicer cleavage siteof the second strand of the DsiRNA/DNA agent (see, e.g., “Left-Extended”DsiRNA agent of FIG. 6).

In certain embodiments, the DsiRNAs of the invention can possessdeoxyribonucleotide residues at sites immediately adjacent to theprojected Dicer enzyme cleavage site(s). For example, in the second,fourth and sixth DsiRNAs shown in FIG. 12, deoxyribonucleotides can befound (starting at the 5′ terminal residue of the first strand asposition 1) at position 22 and sites 3′ of position 22 (e.g., 23, 24,25, etc.). Correspondingly, deoxyribonucleotides can also be found onthe second strand commencing at the nucleotide that is complementary toposition 20 of the first strand, and also at positions on the secondstrand that are located in the 5′ direction of this nucleotide. Thus,certain effective DsiRNAs of the invention possess only 19 duplexedribonucleotides prior to commencement of introduction ofdeoxyribonucleotides within the first strand, second strand, and/or bothstrands of such DsiRNAs. While the preceding statements regardingplacement of deoxyribonucleotides immediately adjacent to a projectedDicer enzyme cleavage site of the DsiRNAs of the invention explicitlycontemplates “right-extended” DsiRNAs of the invention, parallelplacement of deoxyribonucleotides can be performed within“left-extended” DsiRNAs of the invention (e.g., deoxyribonucleotides canbe placed immediately adjacent to the projected Dicer enzyme cleavagesite within “left-extended” DsiRNAs—e.g., immediately 5′ on the sensestrand of the most 5′ projected Dicer cleavage site on the sense strandof such a “left-extended” DsiRNA and/or immediately 3′ on the antisensestrand of the most 3′ projected Dicer cleavage site on the antisensestrand of such a “left-extended” DsiRNA).

As used herein, “duplex” refers to a double helical structure formed bythe interaction of two single stranded nucleic acids. According to thepresent invention, a duplex may contain first and second strands whichare sense and antisense, or which are target and antisense. A duplex istypically formed by the pairwise hydrogen bonding of bases, i.e., “basepairing”, between two single stranded nucleic acids which are orientedantiparallel with respect to each other. Base pairing in duplexesgenerally occurs by Watson-Crick base pairing, e.g., guanine (G) forms abase pair with cytosine (C) in DNA and RNA (thus, the cognate nucleotideof a guanine deoxyribonucleotide is a cytosine deoxyribonucleotide, andvice versa), adenine (A) forms a base pair with thymine (T) in DNA, andadenine (A) forms a base pair with uracil (U) in RNA. Conditions underwhich base pairs can form include physiological or biologically relevantconditions (e.g., intracellular: pH 7.2, 140 mM potassium ion;extracellular pH 7.4, 145 mM sodium ion). Furthermore, duplexes arestabilized by stacking interactions between adjacent nucletotides. Asused herein, a duplex may be established or maintained by base pairingor by stacking interactions. A duplex is formed by two complementarynucleic acid strands, which may be substantially complementary or fullycomplementary (see below).

By “complementary” or “complementarity” is meant that a nucleic acid canform hydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or Hoogsteen base pairing. In reference to thenucleic acid molecules of the present disclosure, the binding freeenergy for a nucleic acid molecule with its complementary sequence issufficient to allow the relevant function of the nucleic acid toproceed, e.g., RNAi activity. Determination of binding free energies fornucleic acid molecules is well known in the art (see, e.g., Turner, etal., CSH Symp. Quant. Biol. LII, pp. 123-133, 1987; Frier, et al., Proc.Nat. Acad. Sci. USA 83:9373-9377, 1986; Turner, et al., J. Am. Chem.Soc. 109:3783-3785, 1987). A percent complementarity indicates thepercentage of contiguous residues in a nucleic acid molecule that canform hydrogen bonds (e.g., Watson-Crick base pairing) with a secondnucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of atotal of 10 nucleotides in the first oligonucleotide being based pairedto a second nucleic acid sequence having 10 nucleotides represents 50%,60%, 70%, 80%, 90%, and 100% complementary, respectively). To determinethat a percent complementarity is of at least a certain percentage, thepercentage of contiguous residues in a nucleic acid molecule that canform hydrogen bonds (e.g., Watson-Crick base pairing) with a secondnucleic acid sequence is calculated and rounded to the nearest wholenumber (e.g., 12, 13, 14, 15, 16, or 17 nucleotides out of a total of 23nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 23 nucleotides represents 52%, 57%, 61%,65%, 70%, and 74%, respectively; and has at least 50%, 50%, 60%, 60%,70%, and 70% complementarity, respectively). As used herein,“substantially complementary” refers to complementarity between thestrands such that they are capable of hybridizing under biologicalconditions. Substantially complementary sequences have 60%, 70%, 80%,90%, 95%, or even 100% complementarity. Additionally, techniques todetermine if two strands are capable of hybridizing under biologicalconditions by examining their nucleotide sequences are well known in theart.

The first and second strands of the agents of the invention (antisenseand sense oligonucleotides) are not required to be completelycomplementary. In one embodiment, the RNA sequence of the antisensestrand contains one or more mismatches or modified nucleotides with baseanalogs. In an exemplary embodiment, such mismatches occur within the 3′region of RNA sequence of the antisense strand (e.g., within the RNAsequence of the antisense strand that is complementary to the target RNAsequence that is positioned 5′ of the projected Argonaute 2 (Ago2) cutsite within the target RNA—see, e.g., FIG. 6 for illustration ofexemplary location of such a mismatch-containing region). In one aspect,about two mismatches or modified nucleotides with base analogs areincorporated within the RNA sequence of the antisense strand that is 3′in the antisense strand of the projected Ago2 cleavage site of thetarget RNA sequence when the target RNA sequence is hybridized.

The use of mismatches or decreased thermodynamic stability (specificallyat or near the 3′-terminal residues of sense/5′-terminal residues of theantisense region of siRNAs) has been proposed to facilitate or favorentry of the antisense strand into RISC (Schwarz et al., 2003; Khvorovaet al., 2003), presumably by affecting some rate-limiting unwindingsteps that occur with entry of the siRNA into RISC. Thus, terminal basecomposition has been included in design algorithms for selecting active21mer siRNA duplexes (Ui-Tei et al., 2004; Reynolds et al., 2004).

In certain embodiments, mismatches (or modified nucleotides with baseanalogs) can be positioned within a parent DsiRNA (optionally a right-or left-extended DsiRNA agent) at or near the predicted 3′-terminus ofthe sense strand of the siRNA projected to be formed following Dicercleavage. In such embodiments, the small end-terminal sequence whichcontains the mismatch(es) will either be left unpaired with theantisense strand (become part of a 3′-overhang) or be cleaved entirelyoff the final 21-mer siRNA. In such embodiments, mismatches in theoriginal (non-Dicer-processed) agent do not persist as mismatches in thefinal RNA component of RISC. It has been found that base mismatches ordestabilization of segments at the 3′-end of the sense strand of Dicersubstrate improved the potency of synthetic duplexes in RNAi, presumablyby facilitating processing by Dicer (Collingwood et al., 2008).

In some embodiments, one or more mismatches are positioned within aDsiRNA agent of the invention (optionally a right- or left-extendedDsiRNA agent) at a location within the region of the antisense strand ofthe DsiRNA agent that hybridizes with the region of the target mRNA thatis positioned 5′ of the predicted Ago2 cleavage site within the targetmRNA (see, e.g., location(s) of mismatches within the agents of FIG. 7).Optionally, two or more mismatches are positioned within the right- orleft-extended DsiRNA agents of the instant invention within thisrelatively 3′ region of the antisense strand that hybridizes to asequence of the target RNA that is positioned 5′ of the projected Ago2cleavage site of the target RNA (were target RNA cleavage to occur).Inclusion of such mismatches within the DsiRNA agents of the instantinvention can allow such agents to exert inhibitory effects thatresemble those of naturally-occurring miRNAs, and optionally can bedirected against not only naturally-occurring miRNA target RNAs (e.g.,3′ UTR regions of target transcripts) but also against RNA sequences forwhich no naturally-occurring antagonistic miRNA is known to exist. Forexample, DsiRNAs of the invention possessing mismatched base pairs whichare designed to resemble and/or function as miRNAs can be synthesized totarget repetitive sequences within genes/transcripts that might not betargeted by naturally-occurring miRNAs (e.g., repeat sequences withinthe Notch protein can be targeted, where individual repeats within Notchcan differ from one another (e.g., be degenerate) at the nucleic acidlevel, but which can be effectively targeted via a miRNA mechanism thatallows for mismatch(es) yet also allows for a more promiscuousinhibitory effect than a corresponding, perfect match siRNA agent). Insuch embodiments, target RNA cleavage may or may not be necessary forthe mismatch-containing DsiRNA agent to exert an inhibitory effect.

In one embodiment, a double stranded nucleic acid molecule of theinvention comprises or functions as a microRNA (miRNA). By “microRNA” or“miRNA” is meant a small double stranded RNA that regulates theexpression of target messenger RNAs either by mRNA cleavage,translational repression/inhibition or heterochromatic silencing (seefor example Ambros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116,281-297; Cullen, 2004, Virus Research., 102, 3-9; He et al., 2004, Nat.Rev. Genet., 5, 522-531; and Ying et al., 2004, Gene, 342, 25-28). Inone embodiment, the microRNA of the invention, has partialcomplementarity (i.e., less than 100% complementarity) between the sensestrand (e.g., first strand) or sense region and the antisense strand(e.g., second strand) or antisense region of the miRNA molecule orbetween the antisense strand or antisense region of the miRNA and acorresponding target nucleic acid molecule (e.g., target mRNA). Forexample, partial complementarity can include various mismatches ornon-base paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches ornon-based paired nucleotides, such as nucleotide bulges) within thedouble stranded nucleic acid molecule structure, which can result inbulges, loops, or overhangs that result between the sense strand orsense region and the antisense strand or antisense region of the miRNAor between the antisense strand or antisense region of the miRNA and acorresponding target nucleic acid molecule.

Single-stranded nucleic acids that base pair over a number of bases aresaid to “hybridize.” Hybridization is typically determined underphysiological or biologically relevant conditions (e.g., intracellular:pH 7.2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion).Hybridization conditions generally contain a monovalent cation andbiologically acceptable buffer and may or may not contain a divalentcation, complex anions, e.g. gluconate from potassium gluconate,uncharged species such as sucrose, and inert polymers to reduce theactivity of water in the sample, e.g. PEG. Such conditions includeconditions under which base pairs can form.

Hybridization is measured by the temperature required to dissociatesingle stranded nucleic acids forming a duplex, i.e., (the meltingtemperature; Tm). Hybridization conditions are also conditions underwhich base pairs can form. Various conditions of stringency can be usedto determine hybridization (see, e.g., Wahl, G. M. and S. L. Berger(1987) Methods Enzymol. 152: 399; Kimmel, A. R. (1987) Methods Enzymol.152:507). Stringent temperature conditions will ordinarily includetemperatures of at least about 30° C., more preferably of at least about37° C., and most preferably of at least about 42° C. The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (Tm) of thehybrid, where Tm is determined according to the following equations. Forhybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+Tbases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs inlength, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41 (% G+C)-(600/N), where N isthe number of bases in the hybrid, and [Na+] is the concentration ofsodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). Forexample, a hybridization determination buffer is shown in Table 1.

TABLE 1 To make 50 final conc. Vender Cat# Lot# m.w./Stock mL solutionNaCl 100 mM Sigma S-5150 41K8934 5M 1 mL KCl 80 mM Sigma P-9541 70K0002 74.55 0.298 g MgCl₂ 8 mM Sigma M-1028 120K8933 1M 0.4 mL sucrose 2% w/vFisher BP220-212 907105 342.3 1 g Tris-HCl 16 mM Fisher BP1757-500 12419 1M 0.8 mL NaH₂PO₄ 1 mM Sigma S-3193 52H-029515 120.0 0.006 g EDTA0.02 mM Sigma E-7889 110K89271 0.5M   2 μL H₂O Sigma W-4502 51K2359 to50 mL pH = 7.0 adjust with at 20° C. HCl

Useful variations on hybridization conditions will be readily apparentto those skilled in the art. Hybridization techniques are well known tothose skilled in the art and are described, for example, in Benton andDavis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad.Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in MolecularBiology, Wiley Interscience, New York, 2001); Berger and Kimmel(Antisense to Molecular Cloning Techniques, 1987, Academic Press, NewYork); and Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, New York.

As used herein, “oligonucleotide strand” is a single stranded nucleicacid molecule. An oligonucleotide may comprise ribonucleotides,deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2′modifications, synthetic base analogs, etc.) or combinations thereof.Such modified oligonucleotides can be preferred over native formsbecause of properties such as, for example, enhanced cellular uptake andincreased stability in the presence of nucleases.

Certain dsNAs of this invention are chimeric dsNAs. “Chimeric dsNAs” or“chimeras”, in the context of this invention, are dsNAs which containtwo or more chemically distinct regions, each made up of at least onenucleotide. These dsNAs typically contain at least one region primarilycomprising ribonucleotides (optionally including modifiedribonucleotides) that form a Dicer substrate siRNA (“DsiRNA”) molecule.This DsiRNA region is covalently attached to a second region comprisingbase paired deoxyribonucleotides (a “dsDNA region”) which confers one ormore beneficial properties (such as, for example, increased efficacy,e.g., increased potency and/or duration of DsiRNA activity, function asa recognition domain or means of targeting a chimeric dsNA to a specificlocation, for example, when administered to cells in culture or to asubject, functioning as an extended region for improved attachment offunctional groups, payloads, detection/detectable moieties, functioningas an extended region that allows for more desirable modificationsand/or improved spacing of such modifications, etc.). This second regioncomprising base paired deoxyribonucleotides may also include modified orsynthetic nucleotides and/or modified or synthetic deoxyribonucleotides.

As used herein, the term “ribonucleotide” encompasses natural andsynthetic, unmodified and modified ribonucleotides. Modificationsinclude changes to the sugar moiety, to the base moiety and/or to thelinkages between ribonucleotides in the oligonucleotide. As used herein,the term “ribonucleotide” specifically excludes a deoxyribonucleotide,which is a nucleotide possessing a single proton group at the 2′ ribosering position.

As used herein, the term “deoxyribonucleotide” encompasses natural andsynthetic, unmodified and modified deoxyribonucleotides. Modificationsinclude changes to the sugar moiety, to the base moiety and/or to thelinkages between deoxyribonucleotide in the oligonucleotide. As usedherein, the term “deoxyribonucleotide” also includes a modifiedribonucleotide that does not permit Dicer cleavage of a dsNA agent,e.g., a 2′-O-methyl ribonucleotide, a phosphorothioate-modifiedribonucleotide residue, etc., that does not permit Dicer cleavage tooccur at a bond of such a residue.

As used herein, the term “PS-NA” refers to a phosphorothioate-modifiednucleotide residue. The term “PS-NA” therefore encompasses bothphosphorothioate-modified ribonucleotides (“PS-RNAs”) andphosphorothioate-modified deoxyribonucleotides (“PS-DNAs”).

In certain embodiments, a chimeric DsiRNA/DNA agent of the inventioncomprises at least one duplex region of at least 23 nucleotides inlength, within which at least 50% of all nucleotides are unmodifiedribonucleotides. As used herein, the term “unmodified ribonucleotide”refers to a ribonucleotide possessing a hydroxyl (—OH) group at the 2′position of the ribose sugar.

In certain embodiments, a chimeric DsiRNA/DNA agent of the inventioncomprises at least one region, located 3′ of the projected Dicercleavage site on the first strand and 5′ of the projected Dicer cleavagesite on the second strand, having a length of at least 2 base pairednucleotides in length, wherein at least 50% of all nucleotides withinthis region of at least 2 base paired nucleotides in length areunmodified deoxyribonucleotides. As used herein, the term “unmodifieddeoxyribonucleotide” refers to a ribonucleotide possessing a singleproton at the 2′ position of the ribose sugar.

As used herein, “antisense strand” refers to a single stranded nucleicacid molecule which has a sequence complementary to that of a targetRNA. When the antisense strand contains modified nucleotides with baseanalogs, it is not necessarily complementary over its entire length, butmust at least hybridize with a target RNA.

As used herein, “sense strand” refers to a single stranded nucleic acidmolecule which has a sequence complementary to that of an antisensestrand. When the antisense strand contains modified nucleotides withbase analogs, the sense strand need not be complementary over the entirelength of the antisense strand, but must at least duplex with theantisense strand.

As used herein, “guide strand” refers to a single stranded nucleic acidmolecule of a dsRNA or dsRNA-containing molecule, which has a sequencesufficiently complementary to that of a target RNA to result in RNAinterference. After cleavage of the dsRNA or dsRNA-containing moleculeby Dicer, a fragment of the guide strand remains associated with RISC,binds a target RNA as a component of the RISC complex, and promotescleavage of a target RNA by RISC. As used herein, the guide strand doesnot necessarily refer to a continuous single stranded nucleic acid andmay comprise a discontinuity, preferably at a site that is cleaved byDicer. A guide strand is an antisense strand.

As used herein, “target RNA” refers to an RNA that would be subject tomodulation guided by the antisense strand, such as targeted cleavage orsteric blockage. The target RNA could be, for example genomic viral RNA,mRNA, a pre-mRNA, or a non-coding RNA. The preferred target is mRNA,such as the mRNA encoding a disease associated protein, such as ApoB,Bcl2, Hif-lalpha, Survivin or a p21 ras, such as Ha. ras, K-ras orN-ras.

As used herein, “passenger strand” refers to an oligonucleotide strandof a dsRNA or dsRNA-containing molecule, which has a sequence that iscomplementary to that of the guide strand. As used herein, the passengerstrand does not necessarily refer to a continuous single strandednucleic acid and may comprise a discontinuity, preferably at a site thatis cleaved by Dicer. A passenger strand is a sense strand.

As used herein, “Dicer” refers to an endoribonuclease in the RNase IIIfamily that cleaves a dsRNA or dsRNA-containing molecule, e.g.,double-stranded RNA (dsRNA) or pre-microRNA (miRNA), intodouble-stranded nucleic acid fragments about 19-25 nucleotides long,usually with a two-base overhang on the 3′ end. With respect to thedsNAs of the invention, the duplex formed by a dsRNA region of a dsNA ofthe invention is recognized by Dicer and is a Dicer substrate on atleast one strand of the duplex. Dicer catalyzes the first step in theRNA interference pathway, which consequently results in the degradationof a target RNA. The protein sequence of human Dicer is provided at theNCBI database under accession number NP_(—)085124, hereby incorporatedby reference.

Dicer “cleavage” is determined as follows (e.g., see Collingwood et al.,Oligonucleotides 18:187-200 (2008)). In a Dicer cleavage assay, RNAduplexes (100 pmol) are incubated in 20 μL of 20 mM Tris pH 8.0, 200 mMNaCl, 2.5 mM MgCl2 with or without 1 unit of recombinant human Dicer(Stratagene, La Jolla, Calif.) at 37° C. for 18-24 hours. Samples aredesalted using a Performa SR 96-well plate (Edge Biosystems,Gaithersburg, Md.). Electrospray-ionization liquid chromatography massspectroscopy (ESI-LCMS) of duplex RNAs pre- and post-treatment withDicer is done using an Oligo HTCS system (Novatia, Princeton, N.J.; Hailet al., 2004), which consists of a ThermoFinnigan TSQ7000, Xcalibur datasystem, ProMass data processing software and Paradigm MS4 HPLC (MichromBioResources, Auburn, Calif.). In this assay, Dicer cleavage occurswhere at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, oreven 100% of the Dicer substrate dsRNA, (i.e., 25-35 bp dsRNA,preferably 26-30 bp dsRNA, optionally extended as described herein) iscleaved to a shorter dsRNA (e.g., 19-23 bp dsRNA, preferably, 21-23 bpdsRNA).

As used herein, “Dicer cleavage site” refers to the sites at which Dicercleaves a dsRNA (e.g., the dsRNA region of a dsNA of the invention).Dicer contains two RNase III domains which typically cleave both thesense and antisense strands of a dsRNA. The average distance between theRNase III domains and the PAZ domain determines the length of the shortdouble-stranded nucleic acid fragments it produces and this distance canvary (Macrae I, et al. (2006). “Structural basis for double-stranded RNAprocessing by Dicer”. Science 311 (5758): 195-8.). As shown in FIG. 1A,Dicer is projected to cleave certain double-stranded nucleic acids ofthe instant invention that possess an antisense strand having a 2nucleotide 3′ overhang at a site between the 21^(st) and 22^(nd)nucleotides removed from the 3′ terminus of the antisense strand, and ata corresponding site between the 21^(st) and 22^(nd) nucleotides removedfrom the 5′ terminus of the sense strand. The projected and/or prevalentDicer cleavage site(s) for dsNA molecules distinct from those depictedin FIG. 1A may be similarly identified via art-recognized methods,including those described in Macrae et al. While the Dicer cleavageevent depicted in FIG. 1A generates a 21 nucleotide siRNA, it is notedthat Dicer cleavage of a dsNA (e.g., DsiRNA) can result in generation ofDicer-processed siRNA lengths of 19 to 23 nucleotides in length. Indeed,in one aspect of the invention that is described in greater detailbelow, a double stranded DNA region is included within a dsNA forpurpose of directing prevalent Dicer excision of a typicallynon-preferred 19mer siRNA.

As used herein, “overhang” refers to unpaired nucleotides, in thecontext of a duplex having two or four free ends at either the 5′terminus or 3′ terminus of a dsNA. In certain embodiments, the overhangis a 3′ or 5′ overhang on the antisense strand or sense strand.

As used herein, “target” refers to any nucleic acid sequence whoseexpression or activity is to be modulated. In particular embodiments,the target refers to an RNA which duplexes to a single stranded nucleicacid that is an antisense strand in a RISC complex. Hybridization of thetarget RNA to the antisense strand results in processing by the RISCcomplex. Consequently, expression of the RNA or proteins encoded by theRNA, e.g., mRNA, is reduced.

As used herein, the term “RNA processing” refers to processingactivities performed by components of the siRNA, miRNA or RNase Hpathways (e.g., Drosha, Dicer, Argonaute2 or other RISCendoribonucleases, and RNaseH), which are described in greater detailbelow (see “RNA Processing” section below). The term is explicitlydistinguished from the post-transcriptional processes of 5′ capping ofRNA and degradation of RNA via non-RISC- or non-RNase H-mediatedprocesses. Such “degradation” of an RNA can take several forms, e.g.deadenylation (removal of a 3′ poly(A) tail), and/or nuclease digestionof part or all of the body of the RNA by any of several endo- orexo-nucleases (e.g., RNase III, RNase P, RNase T1, RNase A (1, 2, 3,4/5), oligonucleotidase, etc.).

As used herein, “reference” is meant a standard or control. As isapparent to one skilled in the art, an appropriate reference is whereonly one element is changed in order to determine the effect of the oneelement.

As used herein, “modified nucleotide” refers to a nucleotide that hasone or more modifications to the nucleoside, the nucleobase, pentosering, or phosphate group. For example, modified nucleotides excluderibonucleotides containing adenosine monophosphate, guanosinemonophosphate, uridine monophosphate, and cytidine monophosphate anddeoxyribonucleotides containing deoxyadenosine monophosphate,deoxyguanosine monophosphate, deoxythymidine monophosphate, anddeoxycytidine monophosphate. Modifications include those naturallyoccuring that result from modification by enzymes that modifynucleotides, such as methyltransferases. Modified nucleotides alsoinclude synthetic or non-naturally occurring nucleotides. Synthetic ornon-naturally occurring modifications in nucleotides include those with2′ modifications, e.g., 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH₂—O-2′-bridge,4′-(CH₂)₂—O-2′-bridge, 2′-LNA, and 2′-O-(N-methylcarbamate) or thosecomprising base analogs. In connection with 2′-modified nucleotides asdescribed for the present disclosure, by “amino” is meant 2′-NH₂ or2′-O—NH₂, which can be modified or unmodified. Such modified groups aredescribed, e.g., in Eckstein et al., U.S. Pat. No. 5,672,695 andMatulic-Adamic et al., U.S. Pat. No. 6,248,878.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

In reference to the nucleic acid molecules of the present disclosure,the modifications may exist in patterns on a strand of the dsNA. As usedherein, “alternating positions” refers to a pattern where every othernucleotide is a modified nucleotide or there is an unmodified nucleotide(e.g., an unmodified ribonucleotide) between every modified nucleotideover a defined length of a strand of the dsNA (e.g., 5′-MNMNMN-3′;3′-MNMNMN-5′; where M is a modified nucleotide and N is an unmodifiednucleotide). The modification pattern starts from the first nucleotideposition at either the 5′ or 3′ terminus according to any of theposition numbering conventions described herein (in certain embodiments,position 1 is designated in reference to the terminal residue of astrand following a projected Dicer cleavage event of a DsiRNA agent ofthe invention; thus, position 1 does not always constitute a 3′ terminalor 5′ terminal residue of a pre-processed agent of the invention). Thepattern of modified nucleotides at alternating positions may run thefull length of the strand, but in certain embodiments includes at least4, 6, 8, 10, 12, 14 nucleotides containing at least 2, 3, 4, 5, 6 or 7modified nucleotides, respectively. As used herein, “alternating pairsof positions” refers to a pattern where two consecutive modifiednucleotides are separated by two consecutive unmodified nucleotides overa defined length of a strand of the dsNA (e.g., 5′-MMNNMMNNMMNN-3′;3′-MMNNMMNNMMNN-5′; where M is a modified nucleotide and N is anunmodified nucleotide). The modification pattern starts from the firstnucleotide position at either the 5′ or 3′ terminus according to any ofthe position numbering conventions described herein. The pattern ofmodified nucleotides at alternating positions may run the full length ofthe strand, but preferably includes at least 8, 12, 16, 20, 24, 28nucleotides containing at least 4, 6, 8, 10, 12 or 14 modifiednucleotides, respectively. It is emphasized that the above modificationpatterns are exemplary and are not intended as limitations on the scopeof the invention.

As used herein, “base analog” refers to a heterocyclic moiety which islocated at the 1′ position of a nucleotide sugar moiety in a modifiednucleotide that can be incorporated into a nucleic acid duplex (or theequivalent position in a nucleotide sugar moiety substitution that canbe incorporated into a nucleic acid duplex). In the dsNAs of theinvention, a base analog is generally either a purine or pyrimidine baseexcluding the common bases guanine (G), cytosine (C), adenine (A),thymine (T), and uracil (U). Base analogs can duplex with other bases orbase analogs in dsRNAs. Base analogs include those useful in thecompounds and methods of the invention., e.g., those disclosed in U.S.Pat. Nos. 5,432,272 and 6,001,983 to Benner and US Patent PublicationNo. 20080213891 to Manoharan, which are herein incorporated byreference. Non-limiting examples of bases include hypoxanthine (I),xanthine (X), 3β-D-ribofuranosyl-(2,6-diaminopyrimidine) (K),3-β-D-ribofuranosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione)(P), iso-cytosine (iso-C), iso-guanine (iso-G),1-β-D-ribofuranosyl-(5-nitroindole),1-β-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-aminopurine,4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) andpyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S),2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole,4-methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methylisocarbostyrilyl, and 3-methyl-7-propynyl isocarbostyrilyl,7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl,9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl,2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl,napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl,tetracenyl, pentacenyl, and structural derivates thereof (Schweitzer etal., J. Org. Chem., 59:7238-7242 (1994); Berger et al., Nucleic AcidsResearch, 28(15):2911-2914 (2000); Moran et al., J. Am. Chem. Soc.,119:2056-2057 (1997); Morales et al., J. Am. Chem. Soc., 121:2323-2324(1999); Guckian et al., J. Am. Chem. Soc., 118:8182-8183 (1996); Moraleset al., J. Am. Chem. Soc., 122(6):1001-1007 (2000); McMinn et al., J.Am. Chem. Soc., 121:11585-11586 (1999); Guckian et al., J. Org. Chem.,63:9652-9656 (1998); Moran et al., Proc. Natl. Acad. Sci.,94:10506-10511 (1997); Das et al., J. Chem. Soc., Perkin Trans.,1:197-206 (2002); Shibata et al., J. Chem. Soc., Perkin Trans., 1:1605-1611 (2001); Wu et al., J. Am. Chem. Soc., 122(32):7621-7632(2000); O'Neill et al., J. Org. Chem., 67:5869-5875 (2002); Chaudhuri etal., J. Am. Chem. Soc., 117:10434-10442 (1995); and U.S. Pat. No.6,218,108.). Base analogs may also be a universal base.

As used herein, “universal base” refers to a heterocyclic moiety locatedat the 1′ position of a nucleotide sugar moiety in a modifiednucleotide, or the equivalent position in a nucleotide sugar moietysubstitution, that, when present in a nucleic acid duplex, can bepositioned opposite more than one type of base without altering thedouble helical structure (e.g., the structure of the phosphatebackbone). Additionally, the universal base does not destroy the abilityof the single stranded nucleic acid in which it resides to duplex to atarget nucleic acid. The ability of a single stranded nucleic acidcontaining a universal base to duplex a target nucleic can be assayed bymethods apparent to one in the art (e.g., UV absorbance, circulardichroism, gel shift, single stranded nuclease sensitivity, etc.).Additionally, conditions under which duplex formation is observed may bevaried to determine duplex stability or formation, e.g., temperature, asmelting temperature (Tm) correlates with the stability of nucleic acidduplexes. Compared to a reference single stranded nucleic acid that isexactly complementary to a target nucleic acid, the single strandednucleic acid containing a universal base forms a duplex with the targetnucleic acid that has a lower Tm than a duplex formed with thecomplementary nucleic acid. However, compared to a reference singlestranded nucleic acid in which the universal base has been replaced witha base to generate a single mismatch, the single stranded nucleic acidcontaining the universal base forms a duplex with the target nucleicacid that has a higher Tm than a duplex formed with the nucleic acidhaving the mismatched base.

Some universal bases are capable of base pairing by forming hydrogenbonds between the universal base and all of the bases guanine (G),cytosine (C), adenine (A), thymine (T), and uracil (U) under base pairforming conditions. A universal base is not a base that forms a basepair with only one single complementary base. In a duplex, a universalbase may form no hydrogen bonds, one hydrogen bond, or more than onehydrogen bond with each of G, C, A, T, and U opposite to it on theopposite strand of a duplex. Preferably, the universal bases does notinteract with the base opposite to it on the opposite strand of aduplex. In a duplex, base pairing between a universal base occurswithout altering the double helical structure of the phosphate backbone.A universal base may also interact with bases in adjacent nucleotides onthe same nucleic acid strand by stacking interactions. Such stackinginteractions stabilize the duplex, especially in situations where theuniversal base does not form any hydrogen bonds with the base positionedopposite to it on the opposite strand of the duplex. Non-limitingexamples of universal-binding nucleotides include inosine,1-β-D-ribofuranosyl-5-nitroindole, and/or1-β-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No. 20070254362to Quay et al.; Van Aerschot et al., An acyclic 5-nitroindazolenucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995Nov. 11; 23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindoleas universal bases in primers for DNA sequencing and PCR. Nucleic AcidsRes. 1995 Jul. 11; 23(13):2361-6; Loakes and Brown, 5-Nitroindole as anuniversal base analogue. Nucleic Acids Res. 1994 Oct. 11;22(20):4039-43).

As used herein, “loop” refers to a structure formed by a single strandof a nucleic acid, in which complementary regions that flank aparticular single stranded nucleotide region hybridize in a way that thesingle stranded nucleotide region between the complementary regions isexcluded from duplex formation or Watson-Crick base pairing. A loop is asingle stranded nucleotide region of any length. Examples of loopsinclude the unpaired nucleotides present in such structures as hairpins,stem loops, or extended loops.

As used herein, “extended loop” in the context of a dsRNA refers to asingle stranded loop and in addition 1, 2, 3, 4, 5, 6 or up to 20 basepairs or duplexes flanking the loop. In an extended loop, nucleotidesthat flank the loop on the 5′ side form a duplex with nucleotides thatflank the loop on the 3′ side. An extended loop may form a hairpin orstem loop.

As used herein, “tetraloop” in the context of a dsRNA refers to a loop(a single stranded region) consisting of four nucleotides that forms astable secondary structure that contributes to the stability of anadjacent Watson-Crick hybridized nucleotides. Without being limited totheory, a tetraloop may stabilize an adjacent Watson-Crick base pair bystacking interactions. In addition, interactions among the fournucleotides in a tetraloop include but are not limited tonon-Watson-Crick base pairing, stacking interactions, hydrogen bonding,and contact interactions (Cheong et al., Nature 1990 Aug. 16;346(6285):680-2; Heus and Pardi, Science 1991 Jul. 12; 253(5016):191-4).A tetraloop confers an increase in the melting temperature (Tm) of anadjacent duplex that is higher than expected from a simple model loopsequence consisting of four random bases. For example, a tetraloop canconfer a melting temperature of at least 55° C. in 10 mM NaHPO₄ to ahairpin comprising a duplex of at least 2 base pairs in length. Atetraloop may contain ribonucleotides, deoxyribonucleotides, modifiednucleotides, and combinations thereof. Examples of RNA tetraloopsinclude the UNCG family of tetraloops (e.g., UUCG), the GNRA family oftetraloops (e.g., GAAA), and the CUUG tetraloop. (Woese et al., ProcNatl Acad Sci USA. 1990 November; 87(21):8467-71; Antao et al., NucleicAcids Res. 1991 Nov. 11; 19(21):5901-5). Examples of DNA tetraloopsinclude the d(GNNA) family of tetraloops (e.g., d(GTTA), the d(GNRA))family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG)family of tetraloops, the d(TNCG) family of tetraloops (e.g., d(TTCG)).(Nakano et al. Biochemistry, 41 (48), 14281-14292, 2002.; SHINJI et al.Nippon Kagakkai Koen Yokoshu VOL. 78th; NO. 2; PAGE. 731 (2000).)

As used herein, “increase” or “enhance” is meant to alter positively byat least 5% compared to a reference in an assay. An alteration may be by5%, 10%, 25%, 30%, 50%, 75%, or even by 100% compared to a reference inan assay. By “enhance Dicer cleavage,” it is meant that the processingof a quantity of a dsRNA or dsRNA-containing molecule by Dicer resultsin more Dicer cleaved dsRNA products, that Dicer cleavage reactionoccurs more quickly compared to the processing of the same quantity of areference dsRNA or dsRNA-containing molecule in an in vivo or in vitroassay of this disclosure, or that Dicer cleavage is directed to cleaveat a specific, preferred site within a dsNA and/or generate higherprevalence of a preferred population of cleavage products (e.g., byinclusion of DNA residues as described herein). In one embodiment,enhanced or increased Dicer cleavage of a dsNA molecule is above thelevel of that observed with an appropriate reference dsNA molecule. Inanother embodiment, enhanced or increased Dicer cleavage of a dsNAmolecule is above the level of that observed with an inactive orattenuated molecule.

As used herein “reduce” is meant to alter negatively by at least 5%compared to a reference in an assay. An alteration may be by 5%, 10%,25%, 30%, 50%, 75%, or even by 100% compared to a reference in an assay.By “reduce expression,” it is meant that the expression of the gene, orlevel of RNA molecules or equivalent RNA molecules encoding one or moreproteins or protein subunits, or level or activity of one or moreproteins or protein subunits encoded by a target gene, is reduced belowthat observed in the absence of the nucleic acid molecules (e.g., dsRNAmolecule or dsRNA-containing molecule) in an in vivo or in vitro assayof this disclosure. In one embodiment, inhibition, down-regulation orreduction with a dsNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with dsNA molecules is belowthat level observed in the presence of, e.g., a dsNA molecule withscrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant disclosure is greater in thepresence of the nucleic acid molecule than in its absence.

As used herein, “cell” is meant to include both prokaryotic (e.g.,bacterial) and eukaryotic (e.g., mammalian or plant) cells. Cells may beof somatic or germ line origin, may be totipotent or pluripotent, andmay be dividing or non-dividing. Cells can also be derived from or cancomprise a gamete or an embryo, a stem cell, or a fully differentiatedcell. Thus, the term “cell” is meant to retain its usual biologicalmeaning and can be present in any organism such as, for example, a bird,a plant, and a mammal, including, for example, a human, a cow, a sheep,an ape, a monkey, a pig, a dog, and a cat. Within certain aspects, theterm “cell” refers specifically to mammalian cells, such as human cells,that contain one or more isolated dsNA molecules of the presentdisclosure. In particular aspects, a cell processes dsRNAs ordsRNA-containing molecules resulting in RNA intereference of targetnucleic acids, and contains proteins and protein complexes required forRNAi, e.g., Dicer and RISC.

As used herein, “animal” is meant a multicellular, eukaryotic organism,including a mammal, particularly a human. The methods of the inventionin general comprise administration of an effective amount of the agentsherein, such as an agent of the structures of formulae herein, to asubject (e.g., animal, human) in need thereof, including a mammal,particularly a human. Such treatment will be suitably administered tosubjects, particularly humans, suffering from, having, susceptible to,or at risk for a disease, or a symptom thereof.

By “pharmaceutically acceptable carrier” is meant, a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant disclosure in the physical location mostsuitable for their desired activity.

The present invention is directed to compositions that comprise both adouble stranded RNA (“dsRNA”) duplex and DNA-containing extendedregion—in most embodiments, a dsDNA duplex—within the same agent, andmethods for preparing them, that are capable of reducing the expressionof target genes in eukaryotic cells. One of the strands of the dsRNAregion contains a region of nucleotide sequence that has a length thatranges from about 15 to about 22 nucleotides that can direct thedestruction of the RNA transcribed from the target gene. The dsDNAduplex region of such an agent is not necessarily complementary to thetarget RNA, and, therefore, in such instances does not enhance targetRNA hybridization of the region of nucleotide sequence capable ofdirecting destruction of a target RNA. Double stranded NAs of theinvention can possess strands that are chemically linked, or can alsopossess an extended loop, optionally comprising a tetraloop, that linksthe first and second strands. In some embodiments, the extended loopcontaining the tetraloop is at the 3′ terminus of the sense strand, atthe 5′ terminus of the antisense strand, or both.

In one embodiment, the dsNA of the invention comprises a double strandedRNA duplex region comprising 18-30 nts (for example, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 and 30 nts) in length.

The DsiRNA/dsDNA agents of the instant invention can enhance thefollowing attributes of such agents relative to DsiRNAs lacking suchdsDNA regions: in vitro efficacy (e.g., potency and duration of effect),in vivo efficacy (e.g., potency, duration of effect, pharmacokinetics,pharmacodynamics, intracellular uptake, reduced toxicity). In certainembodiments, the dsDNA region of the instant invention can optionallyprovide an additional agent (or fragment thereof), such as an aptamer orfragment thereof; a binding site (e.g., a “decoy” binding site) for anative or exogenously introduced moiety capable of binding to dsDNA ineither a non-sequence-selective or sequence-specific manner (e.g., thedsDNA-extended region of an agent of the instant invention can bedesigned to comprise one or more transcription factor recognitionsequences and/or the dsDNA-extended region can provide asequence-specific recognition domain for a probe, marker, etc.).

As used herein, the term “pharmacokinetics” refers to the process bywhich a drug is absorbed, distributed, metabolized, and eliminated bythe body. In certain embodiments of the instant invention, enhancedpharmacokinetics of a DsiRNA/dsDNA agent relative to an appropriatecontrol DsiRNA refers to increased absorption and/or distribution ofsuch an agent, and/or slowed metabolism and/or elimination of such aDsiRNA/dsDNA agent from a subject administered such an agent.

As used herein, the term “pharmacodynamics” refers to the action oreffect of a drug on a living organism. In certain embodiments of theinstant invention, enhanced pharmacodynamics of a DsiRNA/dsDNA agentrelative to an appropriate control DsiRNA refers to an increased (e.g.,more potent or more prolonged) action or effect of a DsiRNA/dsDNA agentupon a subject administered such agent, relative to an appropriatecontrol DsiRNA.

As used herein, the term “stabilization” refers to a state of enhancedpersistence of an agent in a selected environment (e.g., in a cell ororganism). In certain embodiments, the DsiRNA/dsDNA chimeric agents ofthe instant invention exhibit enhanced stability relative to appropriatecontrol DsiRNAs. Such enhanced stability can be achieved via enhancedresistance of such agents to degrading enzymes (e.g., nucleases) orother agents.

DsiRNA Design/Synthesis

It was previously shown that longer dsRNA species of from 25 to about 30nucleotides (DsiRNAs) yield unexpectedly effective RNA inhibitoryresults in terms of potency and duration of action, as compared to19-23mer siRNA agents. Without wishing to be bound by the underlyingtheory of the dsRNA processing mechanism, it is thought that the longerdsRNA species serve as a substrate for the Dicer enzyme in the cytoplasmof a cell. In addition to cleaving the dsNA of the invention intoshorter segments, Dicer is thought to facilitate the incorporation of asingle-stranded cleavage product derived from the cleaved dsNA into theRISC complex that is responsible for the destruction of the cytoplasmicRNA of or derived from the target gene. Prior studies (Rossi et al.,U.S. Patent Application No. 2007/0265220) have shown that thecleavability of a dsRNA species (specifically, a DsiRNA agent) by Dicercorresponds with increased potency and duration of action of the dsRNAspecies. The instant invention, at least in part, provides for design ofRNA inhibitory agents that direct the site of Dicer cleavage, such thatpreferred species of Dicer cleavage products are thereby generated.

A model of DsiRNA processing is presented in FIG. 1A. Briefly, Dicerenzyme binds to a DsiRNA agent, resulting in cleavage of the DsiRNA at aposition 19-23 nucleotides removed from a Dicer PAZ domain-associated 3′overhang sequence of the antisense strand of the DsiRNA agent. ThisDicer cleavage event results in excision of those duplexed nucleic acidspreviously located at the 3′ end of the passenger (sense) strand and 5′end of the guide (antisense) strand. (Cleavage of the DsiRNA shown inFIG. 1A typically yields a 19mer duplex with 2-base overhangs at eachend.) As presently modeled in FIG. 1A, this Dicer cleavage eventgenerates a 21-23 nucleotide guide (antisense) strand capable ofdirecting sequence-specific inhibition of target mRNA as a RISCcomponent.

The first and second oligonucleotides of the DsiRNA agents of theinstant invention are not required to be completely complementary. Infact, in one embodiment, the 3′-terminus of the sense strand containsone or more mismatches. In one aspect, about two mismatches areincorporated at the 3′ terminus of the sense strand. In anotherembodiment, the DsiRNA of the invention is a double stranded RNAmolecule containing two RNA oligonucleotides each of which is anidentical number of nucleotides in the range of 27-35 nucleotides inlength and, when annealed to each other, have blunt ends and a twonucleotide mismatch on the 3′-terminus of the sense strand (the5′-terminus of the antisense strand). The use of mismatches or decreasedthermodynamic stability (specifically at the 3′-sense/5′-antisenseposition) has been proposed to facilitate or favor entry of theantisense strand into RISC (Schwarz et al., 2003; Khvorova et al.,2003), presumably by affecting some rate-limiting unwinding steps thatoccur with entry of the siRNA into RISC. Thus, terminal base compositionhas been included in design algorithms for selecting active 21mer siRNAduplexes (Ui-Tei et al., 2004; Reynolds et al., 2004). With Dicercleavage of the dsRNA region of this embodiment, the small end-terminalsequence which contains the mismatches will either be left unpaired withthe antisense strand (become part of a 3′-overhang) or be cleavedentirely off the final 21-mer siRNA. These specific forms of“mismatches”, therefore, do not persist as mismatches in the final RNAcomponent of RISC. The finding that base mismatches or destabilizationof segments at the 3′-end of the sense strand of Dicer substrateimproved the potency of synthetic duplexes in RNAi, presumably byfacilitating processing by Dicer, was a surprising finding of past worksdescribing the design and use of 25-30mer dsRNAs (also termed “DsiRNAs”herein; Rossi et al., U.S. Patent Application Nos. 2005/0277610,2005/0244858 and 2007/0265220). It is now equally surprising thatDsiRNAs having base-paired deoxyribonucleotides at either passenger(sense) or guide (antisense) strand positions that are predicted to be3′ of the most 3′ Dicer cleavage site of the respective passenger orguide strand are at least equally effective as RNA-RNA duplex-extendedDsiRNA agents. Such agents may also harbor mismatches, with suchmismatches being formed by the antisense strand either in reference to(actual or projected hybridation with) the sequence of the sense strandof the DsiRNA agent, or in reference to the target RNA sequence.Exemplary mismatched or wobble base pairs of agents possessingmismatches are G:A, C:A, C:U, G:G, A:A, C:C, U:U, I:A, I:U and I:C. Basepair strength of such agents can also be lessened via modification ofthe nucleotides of such agents, including, e.g., 2-amino- or 2,6-diaminomodifications of guanine and adenine nucleotides.

Exemplary Structures of DsiRNA Agent Compositions

In one aspect, the present invention provides compositions for RNAinterference (RNAi) that possess one or more base paireddeoxyribonucleotides within a region of a double stranded nucleic acid(dsNA) that is positioned 3′ of a projected sense strand Dicer cleavagesite and correspondingly 5′ of a projected antisense strand Dicercleavage site. The compositions of the invention comprise a dsNA whichis a precursor molecule, i.e., the dsNA of the present invention isprocessed in vivo to produce an active small interfering nucleic acid(siRNA). The dsNA is processed by Dicer to an active siRNA which isincorporated into RISC.

In certain embodiments, the DsiRNA agents of the invention can have anyof the following exemplary structures:

In one such embodiment, the DsiRNA comprises:

wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, and “N”=1 to 50or more, but is optionally 1-15 or, optionally, 1-8. “N*”=0 to 15 ormore, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand.

In a related embodiment, the DsiRNA comprises:

wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, and “N”=1 to 50or more, but is optionally 1-15 or, optionally, 1-8. “N*”=0 to 15 ormore, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand.

In another such embodiment, the DsiRNA comprises:

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “D”=DNA,“Z”=DNA or RNA, and “N”=1 to 50 or more, but is optionally 1-15 or,optionally, 1-8. “N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4,5 or 6. In one embodiment, the top strand is the sense strand, and thebottom strand is the antisense strand. Alternatively, the bottom strandis the sense strand and the top strand is the antisense strand, with2′-O-methyl RNA monomers located at alternating residues along the topstrand, rather than the bottom strand presently depicted in the aboveschematic.

In another such embodiment, the DsiRNA comprises:

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “D”=DNA,“Z”=DNA or RNA, and “N”=1 to 50 or more, but is optionally 1-15 or,optionally, 1-8. “N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4,5 or 6. In one embodiment, the top strand is the sense strand, and thebottom strand is the antisense strand. Alternatively, the bottom strandis the sense strand and the top strand is the antisense strand, with2′-O-methyl RNA monomers located at alternating residues along the topstrand, rather than the bottom strand presently depicted in the aboveschematic.

In another embodiment, the DsiRNA comprises:

wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, “Z”=DNA or RNA,and “N”=1 to 50 or more, but is optionally 1-15 or, optionally, 1-8,where at least one D1_(N) is present in the top strand and is basepaired with a corresponding D2_(N) in the bottom strand. Optionally,D1_(N) and D1_(N+1) are base paired with corresponding D2_(N) andD2_(N+1); D1_(N), D1_(N+1) and D1_(N+2) are base paired withcorresponding D2_(N), D1_(N+1) and D1_(N+2), etc. “N*”=0 to 15 or more,but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the topstrand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand, with 2′-O-methyl RNA monomers located atalternating residues along the top strand, rather than the bottom strandpresently depicted in the above schematic.

In any of the above-depicted structures, the 5′ end of either the sensestrand or antisense strand optionally comprises a phosphate group.

In another embodiment, the DNA:DNA-extended DsiRNA comprises strandshaving equal lengths possessing 1-3 mismatched residues that serve toorient Dicer cleavage (specifically, one or more of positions 1, 2 or 3on the first strand of the DsiRNA, when numbering from the 3′-terminalresidue, are mismatched with corresponding residues of the 5′-terminalregion on the second strand when first and second strands are annealedto one another).

An exemplary DNA:DNA-extended DsiRNA agent with two terminal mismatchedresidues is shown:

wherein “X”=RNA, “M”=Nucleic acid residues (RNA, DNA or non-natural ormodified nucleic acids) that do not base pair (hydrogen bond) withcorresponding “M” residues of otherwise complementary strand whenstrands are annealed, “D”=DNA and “N”=1 to 50 or more, but is optionally1-15 or, optionally, 1-8. “N*”=0 to 15 or more, but is optionally 0, 1,2, 3, 4, 5 or 6. Any of the residues of such agents can optionally be2′-O-methyl RNA monomers—alternating positioning of 2′-O-methyl RNAmonomers that commences from the 3′-terminal residue of the bottom(second) strand, as shown for above asymmetric agents, can also be usedin the above “blunt/fray” DsiRNA agent. In one embodiment, the topstrand (first strand) is the sense strand, and the bottom strand (secondstrand) is the antisense strand. Alternatively, the bottom strand is thesense strand and the top strand is the antisense strand. Modificationand DNA:DNA extension patterns paralleling those shown above forasymmetric/overhang agents can also be incorporated into such“blunt/frayed” agents.

In one embodiment, a length-extended DsiRNA agent is provided thatcomprises deoxyribonucleotides positioned at sites modeled to functionvia specific direction of Dicer cleavage, yet which does not require thepresence of a base-paired deoxyribonucleotide in the dsNA structure. Anexemplary structure for such a molecule is shown:

wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, and “D”=DNA. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand. The above structureis modeled to force Dicer to cleave a minimum of a 21mer duplex as itsprimary post-processing form. In embodiments where the bottom strand ofthe above structure is the antisense strand, the positioning of twodeoxyribonucleotide residues at the ultimate and penultimate residues ofthe 5′ end of the antisense strand is likely to reduce off-targeteffects (as prior studies have shown a 2′-O-methyl modification of atleast the penultimate position from the 5′ terminus of the antisensestrand to reduce off-target effects; see, e.g., US 2007/0223427).

In one embodiment, the DsiRNA comprises:

wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, and “N”=1 to 50or more, but is optionally 1-15 or, optionally, 1-8. “N*”=0 to 15 ormore, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand.

In a related embodiment, the DsiRNA comprises:

wherein “X”=RNA, optionally a 2′-O-methyl RNA monomers “D”=DNA, “N”=1 to50 or more, but is optionally 1-15 or, optionally, 1-8. “N*”=0 to 15 ormore, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand.

In another such embodiment, the DsiRNA comprises:

wherein “X”=RNA, optionally a 2′-O-methyl RNA monomers “D”=DNA, “N”=1 to50 or more, but is optionally 1-15 or, optionally, 1-8. “N*”=0 to 15 ormore, but is optionally 0, 1, 2, 3, 4, 5 or 6. “Z”=DNA or RNA. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

wherein “X”=RNA, “X”=2′-O-methyl RNA, “D”=DNA, “Z”=DNA or RNA, and “N”=1to 50 or more, but is optionally 1-15 or, optionally, 1-8. “N*”=0 to 15or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand, with 2′-O-methyl RNA monomers located atalternating residues along the top strand, rather than the bottom strandpresently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

wherein “X”=RNA, “X”=2′-O-methyl RNA, “D”=DNA, “Z”=DNA or RNA, and “N”=1to 50 or more, but is optionally 1-15 or, optionally, 1-8. “N*”=0 to 15or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. “Y” is an optionaloverhang domain comprised of 0-10 RNA monomers that are optionally2′-O-methyl RNA monomers—in certain embodiments, “Y” is an overhangdomain comprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers. In one embodiment, the top strand is the sense strand, and thebottom strand is the antisense strand. Alternatively, the bottom strandis the sense strand and the top strand is the antisense strand, with2′-O-methyl RNA monomers located at alternating residues along the topstrand, rather than the bottom strand presently depicted in the aboveschematic.

In another embodiment, the DsiRNA comprises:

wherein “X”=RNA, “D”=DNA, “Z”=DNA or RNA, and “N”=1 to 50 or more, butis optionally 1-15 or, optionally, 1-8, where at least one D1_(N) ispresent in the top strand and is base paired with a corresponding D2_(N)in the bottom strand. Optionally, D1_(N) and D1_(N+1) are base pairedwith corresponding D2_(N) and D2_(N+1); D1_(N), D1_(N+1) and D1_(N+2)are base paired with corresponding D2_(N), D1_(N+1) and D1_(N+2), etc.“N*”=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In oneembodiment, the top strand is the sense strand, and the bottom strand isthe antisense strand. Alternatively, the bottom strand is the sensestrand and the top strand is the antisense strand, with 2′-O-methyl RNAmonomers located at alternating residues along the top strand, ratherthan the bottom strand presently depicted in the above schematic.

In a related embodiment. the DsiRNA comprises:

wherein “X”=RNA, “D”=DNA, “Y” is an optional overhang domain comprisedof 0-10 RNA monomers that are optionally 2′-O-methyl RNA monomers—incertain embodiments, “Y” is an overhang domain comprised of 1-4 RNAmonomers that are optionally 2′-O-methyl RNA monomers, and “N”=1 to 50or more, but is optionally 1-15 or, optionally, 1-8, where at least oneD1_(N) is present in the top strand and is base paired with acorresponding D2_(N) in the bottom strand. Optionally, D1_(N) andD1_(N+1) are base paired with corresponding D2_(N) and D2_(N+1); D1_(N),D1_(N+1) and D1_(N+2) are base paired with corresponding D2_(N),D1_(N+1) and D1_(N+2), etc. “N*”=0 to 15 or more, but is optionally 0,1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sensestrand, and the bottom strand is the antisense strand. Alternatively,the bottom strand is the sense strand and the top strand is theantisense strand, with 2′-O-methyl RNA monomers located at alternatingresidues along the top strand, rather than the bottom strand presentlydepicted in the above schematic.

In any of the above-depicted structures, the 5′ end of either the sensestrand or antisense strand optionally comprises a phosphate group.

In another embodiment, the DNA:DNA-extended DsiRNA comprises strandshaving equal lengths possessing 1-3 mismatched residues that serve toorient Dicer cleavage (specifically, one or more of positions 1, 2 or 3on the first strand of the DsiRNA, when numbering from the 3′-terminalresidue, are mismatched with corresponding residues of the 5′-terminalregion on the second strand when first and second strands are annealedto one another). An exemplary DNA:DNA-extended DsiRNA agent with twoterminal mismatched residues is shown:

wherein “X”=RNA, “M”=Nucleic acid residues (RNA, DNA or non-natural ormodified nucleic acids) that do not base pair (hydrogen bond) withcorresponding “M” residues of otherwise complementary strand whenstrands are annealed, “D”=DNA and “N”=1 to 50 or more, but is optionally1-15 or, optionally, 1-8. “N*”=0 to 15 or more, but is optionally 0, 1,2, 3, 4, 5 or 6. Any of the residues of such agents can optionally be2′-O-methyl RNA monomers—alternating positioning of 2′-O-methyl RNAmonomers that commences from the 3′-terminal residue of the bottom(second) strand, as shown for above asymmetric agents, can also be usedin the above “blunt/fray” DsiRNA agent. In one embodiment, the topstrand (first strand) is the sense strand, and the bottom strand (secondstrand) is the antisense strand. Alternatively, the bottom strand is thesense strand and the top strand is the antisense strand. Modificationand DNA:DNA extension patterns paralleling those shown above forasymmetric/overhang agents can also be incorporated into such“blunt/frayed” agents.

In another embodiment, a length-extended DsiRNA agent is provided thatcomprises deoxyribonucleotides positioned at sites modeled to functionvia specific direction of Dicer cleavage, yet which does not require thepresence of a base-paired deoxyribonucleotide in the dsNA structure.Exemplary structures for such a molecule are shown:

wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, and “D”=DNA. “N*”=0 to 15or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand. The above structures are modeled toforce Dicer to cleave a minimum of a 21mer duplex as its primarypost-processing form.

In any of the above embodiments where the bottom strand of the abovestructure is the antisense strand, the positioning of twodeoxyribonucleotide residues at the ultimate and penultimate residues ofthe 5′ end of the antisense strand is likely to reduce off-targeteffects (as prior studies have shown a 2′-O-methyl modification of atleast the penultimate position from the 5′ terminus of the antisensestrand to reduce off-target effects; see, e.g., US 2007/0223427).

The extended DsiRNAs of the invention can carry a broad range ofmodification patterns (e.g., 2′-O-methyl RNA patterns within extendedDsiRNA agents). Certain preferred modification patterns of the secondstrand of the extended DsiRNAs of the invention are presented below—itis noted that while many of the below structures depict modification ofnon-extended DsiRNAs, the skilled artisan will recognize that themodification patterns shown are also readily applied to the full rangeof extended DsiRNA structures described elsewhere herein.

In one embodiment, the DsiRNA comprises:

wherein “X”=RNA, “p”=a phosphate group, “Y” is an overhang domaincomprised of 1-4 RNA monomers that are optionally 2′-O-methyl RNAmonomers, and “D”=DNA. In one embodiment, the top strand is the sensestrand, and the bottom strand is the antisense strand. Alternatively,the bottom strand is the sense strand and the top strand is theantisense strand.

In another embodiment, the DsiRNA comprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand.

In another embodiment, the DsiRNA comprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand.

In further embodiments, the DsiRNA comprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In one embodiment, the DsiRNAcomprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In additional embodiments, the DsiRNA comprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In one embodiment, the DsiRNAcomprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In other embodiments, the DsiRNA comprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In one embodiment, the DsiRNAcomprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In further embodiments, the DsiRNA comprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In one embodiment, the DsiRNAcomprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In additional embodiments, the DsiRNA comprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In one embodiment, the DsiRNAcomprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In other embodiments, the DsiRNA comprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In one embodiment, the DsiRNAcomprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In certain additional embodiments, the DsiRNA comprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In one embodiment, the DsiRNAcomprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In additional embodiments, the DsiRNA comprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In one embodiment, the DsiRNAcomprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In further embodiments, the DsiRNA comprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, “Y” is anoverhang domain comprised of 1-4 RNA monomers that are optionally2′-O-methyl RNA monomers, underlined residues are 2′-O-methyl RNAmonomers, and “D”=DNA. The top strand is the sense strand, and thebottom strand is the antisense strand. In one embodiment, the DsiRNAcomprises:

wherein “X”=RNA, “p”=a phosphate group, “X”=2′-O-methyl RNA, and“D”=DNA. The top strand is the sense strand, and the bottom strand isthe antisense strand.

In another embodiment, the DsiRNA comprises strands having equal lengthspossessing 1-3 mismatched residues that serve to orient Dicer cleavage(specifically, one or more of positions 1, 2 or 3 on the first strand ofthe DsiRNA, when numbering from the 3′-terminal residue, are mismatchedwith corresponding residues of the 5′-terminal region on the secondstrand when first and second strands are annealed to one another). Anexemplary 27mer DsiRNA agent with two terminal mismatched residues isshown:

wherein “X”=RNA, “p”=a phosphate group, “M”=Nucleic acid residues (RNA,DNA or non-natural or modified nucleic acids) that do not base pair(hydrogen bond) with corresponding “M” residues of otherwisecomplementary strand when strands are annealed. Any of the residues ofsuch agents can optionally be 2′-O-methyl RNA monomers—alternatingpositioning of 2′-O-methyl RNA monomers that commences from the3′-terminal residue of the bottom (second) strand, as shown for aboveasymmetric agents, can also be used in the above “blunt/fray” DsiRNAagent. In one embodiment, the top strand is the sense strand, and thebottom strand is the antisense strand. Alternatively, the bottom strandis the sense strand and the top strand is the antisense strand.

As used herein “DsiRNAmm” refers to a DisRNA having a “mismatch tolerantregion” containing one, two, three or four mismatched base pairs of theduplex formed by the sense and antisense strands of the DsiRNA, wheresuch mismatches are positioned within the DsiRNA at a location(s) lyingbetween (and thus not including) the two terminal base pairs of eitherend of the DsiRNA. The mismatched base pairs are located within a“mismatch-tolerant region” which is defined herein with respect to thelocation of the projected Ago2 cut site of the corresponding targetnucleic acid. The mismatch tolerant region is located “upstream of” theprojected Ago2 cut site of the target strand. “Upstream” in this contextwill be understood as the 5′-most portion of the DsiRNAmm duplex, where5′ refers to the orientation of the sense strand of the DsiRNA duplex.Therefore, the mismatch tolerant region is upstream of the base on thesense (passenger) strand that corresponds to the projected Ago2 cut siteof the target nucleic acid (see FIG. 14); alternatively, when referringto the antisense (guide) strand of the DsiRNAmm, the mismatch tolerantregion can also be described as positioned downstream of the base thatis complementary to the projected Ago2 cut site of the target nucleicacid, that is, the 3′-most portion of the antisense strand of theDsiRNAmm (where position 1 of the antisense strand is the 5′ terminalnucleotide of the antisense strand, see FIG. 20).

In one embodiment, for example as depicted in FIG. 33, the mismatchtolerant region is positioned between and including base pairs 3-9 whennumbered from the nucleotide starting at the 5′ end of the sense strandof the duplex. Therefore, a DsiRNAmm of the invention possesses a singlemismatched base pair at any one of positions 3, 4, 5, 6, 7, 8 or 9 ofthe sense strand of a right-hand extended DsiRNA (where position 1 isthe 5′ terminal nucleotide of the sense strand and position 9 is thenucleotide residue of the sense strand that is immediately 5′ of theprojected Ago2 cut site of the target RNA sequence corresponding to thesense strand sequence). In certain embodiments, for a DsiRNAmm thatpossesses a mismatched base pair nucleotide at any of positions 3, 4, 5,6, 7, 8 or 9 of the sense strand, the corresponding mismatched base pairnucleotide of the antisense strand not only forms a mismatched base pairwith the DsiRNAmm sense strand sequence, but also forms a mismatchedbase pair with a DsiRNAmm target RNA sequence (thus, complementaritybetween the antisense strand sequence and the sense strand sequence isdisrupted at the mismatched base pair within the DsiRNAmm, andcomplementarity is similarly disrupted between the antisense strandsequence of the DsiRNAmm and the target RNA sequence). In alternativeembodiments, the mismatch base pair nucleotide of the antisense strandof a DsiRNAmm only form a mismatched base pair with a correspondingnucleotide of the sense strand sequence of the DsiRNAmm, yet base pairswith its corresponding target RNA sequence nucleotide (thus,complementarity between the antisense strand sequence and the sensestrand sequence is disrupted at the mismatched base pair within theDsiRNAmm, yet complementarity is maintained between the antisense strandsequence of the DsiRNAmm and the target RNA sequence).

A DsiRNAmm of the invention that possesses a single mismatched base pairwithin the mismatch-tolerant region (mismatch region) as described above(e.g., a DsiRNAmm harboring a mismatched nucleotide residue at any oneof positions 3, 4, 5, 6, 7, 8 or 9 of the sense strand) can furtherinclude one, two or even three additional mismatched base pairs. Inpreferred embodiments, these one, two or three additional mismatchedbase pairs of the DsiRNAmm occur at position(s) 3, 4, 5, 6, 7, 8 and/or9 of the sense strand (and at corresponding residues of the antisensestrand). In one embodiment where one additional mismatched base pair ispresent within a DsiRNAmm, the two mismatched base pairs of the sensestrand can occur, e.g., at nucleotides of both position 4 and position 6of the sense strand (with mismatch also occurring at correspondingnucleotide residues of the antisense strand).

In DsiRNAmm agents possessing two mismatched base pairs, mismatches canoccur consecutively (e.g., at consecutive positions along the sensestrand nucleotide sequence). Alternatively, nucleotides of the sensestrand that form mismatched base pairs with the antisense strandsequence can be interspersed by nucleotides that base pair with theantisense strand sequence (e.g., for a DsiRNAmm possessing mismatchednucleotides at positions 3 and 6, but not at positions 4 and 5, themismatched residues of sense strand positions 3 and 6 are interspersedby two nucleotides that form matched base pairs with correspondingresidues of the antisense strand). For example, two residues of thesense strand (located within the mismatch-tolerant region of the sensestrand) that form mismatched base pairs with the corresponding antisensestrand sequence can occur with zero, one, two, three, four or fivematched base pairs located between these mismatched base pairs.

For certain DsiRNAmm agents possessing three mismatched base pairs,mismatches can occur consecutively (e.g., in a triplet along the sensestrand nucleotide sequence). Alternatively, nucleotides of the sensestrand that form mismatched base pairs with the antisense strandsequence can be interspersed by nucleotides that form matched base pairswith the antisense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 3, 4 and 8, but not at positions 5,6 and 7, the mismatched residues of sense strand positions 3 and 4 areadjacent to one another, while the mismatched residues of sense strandpositions 4 and 8 are interspersed by three nucleotides that formmatched base pairs with corresponding residues of the antisense strand).For example, three residues of the sense strand (located within themismatch-tolerant region of the sense strand) that form mismatched basepairs with the corresponding antisense strand sequence can occur withzero, one, two, three or four matched base pairs located between any twoof these mismatched base pairs.

For certain DsiRNAmm agents possessing four mismatched base pairs,mismatches can occur consecutively (e.g., in a quadruplet along thesense strand nucleotide sequence). Alternatively, nucleotides of thesense strand that form mismatched base pairs with the antisense strandsequence can be interspersed by nucleotides that form matched base pairswith the antisense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 3, 5, 7 and 8, but not at positions4 and 6, the mismatched residues of sense strand positions 7 and 8 areadjacent to one another, while the mismatched residues of sense strandpositions 3 and 5 are interspersed by one nucleotide that forms amatched base pair with the corresponding residue of the antisensestrand—similarly, the mismatched residues of sense strand positions 5and 7 are also interspersed by one nucleotide that forms a matched basepair with the corresponding residue of the antisense strand). Forexample, four residues of the sense strand (located within themismatch-tolerant region of the sense strand) that form mismatched basepairs with the corresponding antisense strand sequence can occur withzero, one, two or three matched base pairs located between any two ofthese mismatched base pairs.

In another embodiment, for example as depicted in FIG. 39, a DsiRNAmm ofthe invention comprises a mismatch tolerant region which possesses asingle mismatched base pair nucleotide at any one of positions 13, 14,15, 16, 17, 18, 19, 20 or 21 of the antisense strand of a left-handextended DsiRNA (where position 1 is the 5′ terminal nucleotide of theantisense strand and position 13 is the nucleotide residue of theantisense strand that is immediately 3′ (downstream) in the antisensestrand of the projected Ago2 cut site of the target RNA sequencesufficiently complementary to the antisense strand sequence). In certainembodiments, for a DsiRNAmm that possesses a mismatched base pairnucleotide at any of positions 13, 14, 15, 16, 17, 18, 19, 20 or 21 ofthe antisense strand with respect to the sense strand of the DsiRNAmm,the mismatched base pair nucleotide of the antisense strand not onlyforms a mismatched base pair with the DsiRNAmm sense strand sequence,but also forms a mismatched base pair with a DsiRNAmm target RNAsequence (thus, complementarity between the antisense strand sequenceand the sense strand sequence is disrupted at the mismatched base pairwithin the DsiRNAmm, and complementarity is similarly disrupted betweenthe antisense strand sequence of the DsiRNAmm and the target RNAsequence). In alternative embodiments, the mismatch base pair nucleotideof the antisense strand of a DsiRNAmm only forms a mismatched base pairwith a corresponding nucleotide of the sense strand sequence of theDsiRNAmm, yet base pairs with its corresponding target RNA sequencenucleotide (thus, complementarity between the antisense strand sequenceand the sense strand sequence is disrupted at the mismatched base pairwithin the DsiRNAmm, yet complementarity is maintained between theantisense strand sequence of the DsiRNAmm and the target RNA sequence).

A DsiRNAmm of the invention that possesses a single mismatched base pairwithin the mismatch-tolerant region as described above (e.g., a DsiRNAmmharboring a mismatched nucleotide residue at positions 13, 14, 15, 16,17, 18, 19, 20 or 21 of the antisense strand) can further include one,two or even three additional mismatched base pairs. In preferredembodiments, these one, two or three additional mismatched base pairs ofthe DsiRNAmm occur at position(s) 13, 14, 15, 16, 17, 18, 19, 20 and/or21 of the antisense strand (and at corresponding residues of the sensestrand). In one embodiment where one additional mismatched base pair ispresent within a DsiRNAmm, the two mismatched base pairs of theantisense strand can occur, e.g., at nucleotides of both position 14 andposition 18 of the antisense strand (with mismatch also occurring atcorresponding nucleotide residues of the sense strand).

In DsiRNAmm agents possessing two mismatched base pairs, mismatches canoccur consecutively (e.g., at consecutive positions along the antisensestrand nucleotide sequence). Alternatively, nucleotides of the antisensestrand that form mismatched base pairs with the sense strand sequencecan be interspersed by nucleotides that base pair with the sense strandsequence (e.g., for a DsiRNAmm possessing mismatched nucleotides atpositions 13 and 16, but not at positions 14 and 15, the mismatchedresidues of antisense strand positions 13 and 16 are interspersed by twonucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, two residues of the antisense strand(located within the mismatch-tolerant region of the sense strand) thatform mismatched base pairs with the corresponding sense strand sequencecan occur with zero, one, two, three, four, five, six or seven matchedbase pairs located between these mismatched base pairs.

For certain DsiRNAmm agents possessing three mismatched base pairs,mismatches can occur consecutively (e.g., in a triplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 13, 14 and 18, but not at positions15, 16 and 17, the mismatched residues of antisense strand positions 13and 14 are adjacent to one another, while the mismatched residues ofantisense strand positions 14 and 18 are interspersed by threenucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, three residues of the antisense strand(located within the mismatch-tolerant region of the antisense strand)that form mismatched base pairs with the corresponding sense strandsequence can occur with zero, one, two, three, four, five or six matchedbase pairs located between any two of these mismatched base pairs.

For certain DsiRNAmm agents possessing four mismatched base pairs,mismatches can occur consecutively (e.g., in a quadruplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 13, 15, 17 and 18, but not atpositions 14 and 16, the mismatched residues of antisense strandpositions 17 and 18 are adjacent to one another, while the mismatchedresidues of antisense strand positions 13 and 15 are interspersed by onenucleotide that forms a matched base pair with the corresponding residueof the sense strand—similarly, the mismatched residues of antisensestrand positions 15 and 17 are also interspersed by one nucleotide thatforms a matched base pair with the corresponding residue of the sensestrand). For example, four residues of the antisense strand (locatedwithin the mismatch-tolerant region of the antisense strand) that formmismatched base pairs with the corresponding sense strand sequence canoccur with zero, one, two, three, four or five matched base pairslocated between any two of these mismatched base pairs.

In a further embodiment, for example as depicted in FIG. 40, a DsiRNAmmof the invention possesses a single mismatched base pair nucleotide atany one of positions 11, 12, 13, 14, 15, 16, 17, 18 or 19 of theantisense strand of a left-hand extended DsiRNA (where position 1 is the5′ terminal nucleotide of the antisense strand and position 11 is thenucleotide residue of the antisense strand that is immediately 3′(downstream) in the antisense strand of the projected Ago2 cut site ofthe target RNA sequence sufficiently complementary to the antisensestrand sequence). In certain embodiments, for a DsiRNAmm that possessesa mismatched base pair nucleotide at any of positions 11, 12, 13, 14,15, 16, 17, 18 or 19 of the antisense strand with respect to the sensestrand of the DsiRNAmm, the mismatched base pair nucleotide of theantisense strand not only forms a mismatched base pair with the DsiRNAmmsense strand sequence, but also forms a mismatched base pair with aDsiRNAmm target RNA sequence (thus, complementarity between theantisense strand sequence and the sense strand sequence is disrupted atthe mismatched base pair within the DsiRNAmm, and complementarity issimilarly disrupted between the antisense strand sequence of theDsiRNAmm and the target RNA sequence). In alternative embodiments, themismatch base pair nucleotide of the antisense strand of a DsiRNAmm onlyforms a mismatched base pair with a corresponding nucleotide of thesense strand sequence of the DsiRNAmm, yet this same antisense strandnucleotide base pairs with its corresponding target RNA sequencenucleotide (thus, complementarity between the antisense strand sequenceand the sense strand sequence is disrupted at the mismatched base pairwithin the DsiRNAmm, yet complementarity is maintained between theantisense strand sequence of the DsiRNAmm and the target RNA sequence).

A DsiRNAmm of the invention that possesses a single mismatched base pairwithin the mismatch-tolerant region as described above (e.g., a DsiRNAmmharboring a mismatched nucleotide residue at positions 11, 12, 13, 14,15, 16, 17, 18 or 19 of the antisense strand) can further include one,two or even three additional mismatched base pairs. In preferredembodiments, these one, two or three additional mismatched base pairs ofthe DsiRNAmm occur at position(s) 11, 12, 13, 14, 15, 16, 17, 18 and/or19 of the antisense strand (and at corresponding residues of the sensestrand). In one embodiment where one additional mismatched base pair ispresent within a DsiRNAmm, the two mismatched base pairs of theantisense strand can occur, e.g., at nucleotides of both position 14 andposition 18 of the antisense strand (with mismatch also occurring atcorresponding nucleotide residues of the sense strand).

In DsiRNAmm agents possessing two mismatched base pairs, mismatches canoccur consecutively (e.g., at consecutive positions along the antisensestrand nucleotide sequence). Alternatively, nucleotides of the antisensestrand that form mismatched base pairs with the sense strand sequencecan be interspersed by nucleotides that base pair with the sense strandsequence (e.g., for a DsiRNAmm possessing mismatched nucleotides atpositions 12 and 15, but not at positions 13 and 14, the mismatchedresidues of antisense strand positions 12 and 15 are interspersed by twonucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, two residues of the antisense strand(located within the mismatch-tolerant region of the sense strand) thatform mismatched base pairs with the corresponding sense strand sequencecan occur with zero, one, two, three, four, five, six or seven matchedbase pairs located between these mismatched base pairs.

For certain DsiRNAmm agents possessing three mismatched base pairs,mismatches can occur consecutively (e.g., in a triplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 13, 14 and 18, but not at positions15, 16 and 17, the mismatched residues of antisense strand positions 13and 14 are adjacent to one another, while the mismatched residues ofantisense strand positions 14 and 18 are interspersed by threenucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, three residues of the antisense strand(located within the mismatch-tolerant region of the antisense strand)that form mismatched base pairs with the corresponding sense strandsequence can occur with zero, one, two, three, four, five or six matchedbase pairs located between any two of these mismatched base pairs.

For certain DsiRNAmm agents possessing four mismatched base pairs,mismatches can occur consecutively (e.g., in a quadruplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 13, 15, 17 and 18, but not atpositions 14 and 16, the mismatched residues of antisense strandpositions 17 and 18 are adjacent to one another, while the mismatchedresidues of antisense strand positions 13 and 15 are interspersed by onenucleotide that forms a matched base pair with the corresponding residueof the sense strand—similarly, the mismatched residues of antisensestrand positions 15 and 17 are also interspersed by one nucleotide thatforms a matched base pair with the corresponding residue of the sensestrand). For example, four residues of the antisense strand (locatedwithin the mismatch-tolerant region of the antisense strand) that formmismatched base pairs with the corresponding sense strand sequence canoccur with zero, one, two, three, four or five matched base pairslocated between any two of these mismatched base pairs.

In an additional embodiment, for example as depicted in FIG. 41, aDsiRNAmm of the invention possesses a single mismatched base pairnucleotide at any one of positions 15, 16, 17, 18, 19, 20, 21, 22 or 23of the antisense strand of a left-hand extended DsiRNA (where position 1is the 5′ terminal nucleotide of the antisense strand and position 15 isthe nucleotide residue of the antisense strand that is immediately 3′(downstream) in the antisense strand of the projected Ago2 cut site ofthe target RNA sequence sufficiently complementary to the antisensestrand sequence). In certain embodiments, for a DsiRNAmm that possessesa mismatched base pair nucleotide at any of positions 15, 16, 17, 18,19, 20, 21, 22 or 23 of the antisense strand with respect to the sensestrand of the DsiRNAmm, the mismatched base pair nucleotide of theantisense strand not only forms a mismatched base pair with the DsiRNAmmsense strand sequence, but also forms a mismatched base pair with aDsiRNAmm target RNA sequence (thus, complementarity between theantisense strand sequence and the sense strand sequence is disrupted atthe mismatched base pair within the DsiRNAmm, and complementarity issimilarly disrupted between the antisense strand sequence of theDsiRNAmm and the target RNA sequence). In alternative embodiments, themismatch base pair nucleotide of the antisense strand of a DsiRNAmm onlyforms a mismatched base pair with a corresponding nucleotide of thesense strand sequence of the DsiRNAmm, yet this same antisense strandnucleotide base pairs with its corresponding target RNA sequencenucleotide (thus, complementarity between the antisense strand sequenceand the sense strand sequence is disrupted at the mismatched base pairwithin the DsiRNAmm, yet complementarity is maintained between theantisense strand sequence of the DsiRNAmm and the target RNA sequence).

A DsiRNAmm of the invention that possesses a single mismatched base pairwithin the mismatch-tolerant region as described above (e.g., a DsiRNAmmharboring a mismatched nucleotide residue at positions 15, 16, 17, 18,19, 20, 21, 22 or 23 of the antisense strand) can further include one,two or even three additional mismatched base pairs. In preferredembodiments, these one, two or three additional mismatched base pairs ofthe DsiRNAmm occur at position(s) 15, 16, 17, 18, 19, 20, 21, 22 and/or23 of the antisense strand (and at corresponding residues of the sensestrand). In one embodiment where one additional mismatched base pair ispresent within a DsiRNAmm, the two mismatched base pairs of theantisense strand can occur, e.g., at nucleotides of both position 16 andposition 20 of the antisense strand (with mismatch also occurring atcorresponding nucleotide residues of the sense strand).

In DsiRNAmm agents possessing two mismatched base pairs, mismatches canoccur consecutively (e.g., at consecutive positions along the antisensestrand nucleotide sequence). Alternatively, nucleotides of the antisensestrand that form mismatched base pairs with the sense strand sequencecan be interspersed by nucleotides that base pair with the sense strandsequence (e.g., for a DsiRNAmm possessing mismatched nucleotides atpositions 16 and 20, but not at positions 17, 18 and 19, the mismatchedresidues of antisense strand positions 16 and 20 are interspersed bythree nucleotides that form matched base pairs with correspondingresidues of the sense strand). For example, two residues of theantisense strand (located within the mismatch-tolerant region of thesense strand) that form mismatched base pairs with the correspondingsense strand sequence can occur with zero, one, two, three, four, five,six or seven matched base pairs located between these mismatched basepairs.

For certain DsiRNAmm agents possessing three mismatched base pairs,mismatches can occur consecutively (e.g., in a triplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 16, 17 and 21, but not at positions18, 19 and 20, the mismatched residues of antisense strand positions 16and 17 are adjacent to one another, while the mismatched residues ofantisense strand positions 17 and 21 are interspersed by threenucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, three residues of the antisense strand(located within the mismatch-tolerant region of the antisense strand)that form mismatched base pairs with the corresponding sense strandsequence can occur with zero, one, two, three, four, five or six matchedbase pairs located between any two of these mismatched base pairs.

For certain DsiRNAmm agents possessing four mismatched base pairs,mismatches can occur consecutively (e.g., in a quadruplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 17, 19, 21 and 22, but not atpositions 18 and 20, the mismatched residues of antisense strandpositions 21 and 22 are adjacent to one another, while the mismatchedresidues of antisense strand positions 17 and 19 are interspersed by onenucleotide that forms a matched base pair with the corresponding residueof the sense strand—similarly, the mismatched residues of antisensestrand positions 19 and 21 are also interspersed by one nucleotide thatforms a matched base pair with the corresponding residue of the sensestrand). For example, four residues of the antisense strand (locatedwithin the mismatch-tolerant region of the antisense strand) that formmismatched base pairs with the corresponding sense strand sequence canoccur with zero, one, two, three, four or five matched base pairslocated between any two of these mismatched base pairs.

For reasons of clarity, the location(s) of mismatched nucleotideresidues within the above DsiRNAmm agents are numbered in reference tothe 5′ terminal residue of either sense or antisense strands of theDsiRNAmm. As noted for the different left-extended DsiRNAmm agentsexemplified in FIGS. 20, 21 and 22, the numbering of positions locatedwithin the mismatch-tolerant region (mismatch region) of the antisensestrand can shift with variations in the proximity of the 5′ terminus ofthe antisense strand to the projected Ago2 cleavage site. Thus, thelocation(s) of preferred mismatch sites within either antisense strandor sense strand can also be identified as the permissible proximity ofsuch mismatches to the projected Ago2 cut site. Accordingly, in onepreferred embodiment, the position of a mismatch nucleotide of the sensestrand of a DsiRNAmm is the nucleotide residue of the sense strand thatis located immediately 5′ (upstream) of the projected Ago2 cleavage siteof the corresponding target RNA sequence. In other preferredembodiments, a mismatch nucleotide of the sense strand of a DsiRNAmm ispositioned at the nucleotide residue of the sense strand that is locatedtwo nucleotides 5′ (upstream) of the projected Ago2 cleavage site, threenucleotides 5′ (upstream) of the projected Ago2 cleavage site, fournucleotides 5′ (upstream) of the projected Ago2 cleavage site, fivenucleotides 5′ (upstream) of the projected Ago2 cleavage site, sixnucleotides 5′ (upstream) of the projected Ago2 cleavage site, sevennucleotides 5′ (upstream) of the projected Ago2 cleavage site, eightnucleotides 5′ (upstream) of the projected Ago2 cleavage site, or ninenucleotides 5′ (upstream) of the projected Ago2 cleavage site.

Exemplary single mismatch-containing 25/27mer DsiRNAs (DsiRNAmm) includethe following structures (such mismatch-containing structures may alsobe incorporated into other exemplary DsiRNA structures shown herein).

wherein “X”=RNA, “D”=DNA, “p”=a phosphate group, “M”=Nucleic acidresidues (RNA, DNA or non-natural or modified nucleic acids) that do notbase pair (hydrogen bond) with corresponding “M” residues of otherwisecomplementary strand when strands are annealed. Any of the residues ofsuch agents can optionally be 2′-O-methyl RNA monomers—alternatingpositioning of 2′-O-methyl RNA monomers that commences from the3′-terminal residue of the bottom (second) strand, as shown above, canalso be used in the above DsiRNAmm agents. For the above mismatchstructures, the top strand is the sense strand, and the bottom strand isthe antisense strand.

In certain embodiments, a DsiRNA of the invention can contain mismatchesthat exist in reference to the target RNA sequence yet do notnecessarily exist as mismatched base pairs within the two strands of theDsiRNA—thus, a DsiRNA can possess perfect complementarity between firstand second strands of a DsiRNA, yet still possess mismatched residues inreference to a target RNA (which, in certain embodiments, may beadvantageous in promoting efficacy and/or potency and/or duration ofeffect). In certain embodiments, where mismatches occur betweenantisense strand and target RNA sequence, the position of a mismatch islocated within the antisense strand at a position(s) that corresponds toa sequence of the sense strand located 5′ of the projected Ago2 cut siteof the target region—e.g., antisense strand residue(s) positioned withinthe antisense strand to the 3′ of the antisense residue which iscomplementary to the projected Ago2 cut site of the target sequence(such region is indicated within, e.g., FIG. 33 as a “mismatch region”,which is distinct from the projected “seed region” of such DsiRNAs).

Exemplary 25/27mer DsiRNAs that harbor a single mismatched residue inreference to target sequences include the following preferredstructures.

wherein “X”=RNA, “D”=DNA, “p”=a phosphate group, “E”=Nucleic acidresidues (RNA, DNA or non-natural or modified nucleic acids) that do notbase pair (hydrogen bond) with corresponding “A” RNA residues ofotherwise complementary (target) strand when strands are annealed, yetoptionally do base pair with corresponding “B” residues (“B” residuesare also RNA, DNA or non-natural or modified nucleic acids). Any of theresidues of such agents can optionally be 2′-O-methyl RNA monomers—e.g.,alternating positioning of 2′-O-methyl RNA monomers that commences fromthe 3′-terminal residue of the bottom (second) strand, as shown above,or other patterns of 2′-O-methyl and/or other modifications as describedherein can also be used in the above DsiRNA agents.

In addition to the above-exemplified structures, DsiRNAs of theinvention can also possess one, two or three additional residues thatform further mismatches with the target RNA sequence. Such mismatchescan be consecutive, or can be interspersed by nucleotides that formmatched base pairs with the target RNA sequence. Where interspersed bynucleotides that form matched base pairs, mismatched residues can bespaced apart from each other within a single strand at an interval ofone, two, three, four, five, six, seven or even eight base pairednucleotides between such mismatch-forming residues.

As for the above-described DsiRNAmm agents, a preferred location withinDsiRNAs for antisense strand nucleotides that form mismatched base pairswith target RNA sequence (yet may or may not form mismatches withcorresponding sense strand nucleotides) is within the antisense strandregion that is located 3′ (downstream) of the antisense strand sequencewhich is complementary to the projected Ago2 cut site of the DsiRNA(e.g., in FIG. 39, the region of the antisense strand which is labeledas the “mismatch region” is preferred for mismatch-forming residues andhappens to be located at positions 13-21 of the antisense strand for theagents shown in FIG. 39). Thus, in one preferred embodiment, theposition of a mismatch nucleotide (in relation to the target RNAsequence) of the antisense strand of a DsiRNAmm is the nucleotideresidue of the antisense strand that is located immediately 3′(downstream) within the antisense strand sequence of the projected Ago2cleavage site of the corresponding target RNA sequence. In otherpreferred embodiments, a mismatch nucleotide of the antisense strand ofa DsiRNAmm (in relation to the target RNA sequence) is positioned at thenucleotide residue of the antisense strand that is located twonucleotides 3′ (downstream) of the corresponding projected Ago2 cleavagesite, three nucleotides 3′ (downstream) of the corresponding projectedAgo2 cleavage site, four nucleotides 3′ (downstream) of thecorresponding projected Ago2 cleavage site, five nucleotides 3′(downstream) of the corresponding projected Ago2 cleavage site, sixnucleotides 3′ (downstream) of the projected Ago2 cleavage site, sevennucleotides 3′ (downstream) of the projected Ago2 cleavage site, eightnucleotides 3′ (downstream) of the projected Ago2 cleavage site, or ninenucleotides 3′ (downstream) of the projected Ago2 cleavage site.

In DsiRNA agents possessing two mismatch-forming nucleotides of theantisense strand (where mismatch-forming nucleotides are mismatchforming in relation to target RNA sequence), mismatches can occurconsecutively (e.g., at consecutive positions along the antisense strandnucleotide sequence). Alternatively, nucleotides of the antisense strandthat form mismatched base pairs with the target RNA sequence can beinterspersed by nucleotides that base pair with the target RNA sequence(e.g., for a DsiRNA possessing mismatch-forming nucleotides at positions13 and 16 (starting from the 5′ terminus (position 1) of the antisensestrand of the structure shown in FIG. 39), but not at positions 14 and15, the mismatched residues of sense strand positions 13 and 16 areinterspersed by two nucleotides that form matched base pairs withcorresponding residues of the target RNA sequence). For example, tworesidues of the antisense strand (located within the mismatch-tolerantregion of the antisense strand) that form mismatched base pairs with thecorresponding target RNA sequence can occur with zero, one, two, three,four or five matched base pairs (with respect to target RNA sequence)located between these mismatch-forming base pairs.

For certain DsiRNAs possessing three mismatch-forming base pairs(mismatch-forming with respect to target RNA sequence), mismatch-formingnucleotides can occur consecutively (e.g., in a triplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the target RNAsequence can be interspersed by nucleotides that form matched base pairswith the target RNA sequence (e.g., for a DsiRNA possessing mismatchednucleotides at positions 13, 14 and 18, but not at positions 15, 16 and17, the mismatch-forming residues of antisense strand positions 13 and14 are adjacent to one another, while the mismatch-forming residues ofantisense strand positions 14 and 18 are interspersed by threenucleotides that form matched base pairs with corresponding residues ofthe target RNA). For example, three residues of the antisense strand(located within the mismatch-tolerant region of the antisense strand)that form mismatched base pairs with the corresponding target RNAsequence can occur with zero, one, two, three or four matched base pairslocated between any two of these mismatch-forming base pairs.

For certain DsiRNAs possessing four mismatch-forming base pairs(mismatch-forming with respect to target RNA sequence), mismatch-formingnucleotides can occur consecutively (e.g., in a quadruplet along thesense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the target RNAsequence can be interspersed by nucleotides that form matched base pairswith the target RNA sequence (e.g., for a DsiRNA possessingmismatch-forming nucleotides at positions 13, 15, 17 and 18, but not atpositions 14 and 16, the mismatch-forming residues of antisense strandpositions 17 and 18 are adjacent to one another, while themismatch-forming residues of antisense strand positions 13 and 15 areinterspersed by one nucleotide that forms a matched base pair with thecorresponding residue of the target RNA sequence—similarly, themismatch-forming residues of antisense strand positions 15 and 17 arealso interspersed by one nucleotide that forms a matched base pair withthe corresponding residue of the target RNA sequence). For example, fourresidues of the antisense strand (located within the mismatch-tolerantregion of the antisense strand) that form mismatched base pairs with thecorresponding target RNA sequence can occur with zero, one, two or threematched base pairs located between any two of these mismatch-formingbase pairs.

The above DsiRNAmm and other DsiRNA structures are described in order toexemplify certain structures of DsiRNAmm and DsiRNA agents. Design ofthe above DsiRNAmm and DsiRNA structures can be adapted to generate,e.g., DsiRNAmm forms of a DNA-extended (“DNA handle”) DsiRNA agent showninfra (including, e.g., design of mismatch-containing DsiRNAmm agents asshown in FIGS. 14-16 and 20-22). As exemplified above, DsiRNAs can alsobe designed that possess single mismatches (or two, three or fourmismatches) between the antisense strand of the DsiRNA and a targetsequence, yet optionally can retain perfect complementarity betweensense and antisense strand sequences of a DsiRNA.

It is further noted that the DsiRNA agents exemplified infra can alsopossess insertion/deletion (in/del) structures within theirdouble-stranded and/or target RNA-aligned structures. Accordingly, theDsiRNAs of the invention can be designed to possess in/del variationsin, e.g., antisense strand sequence as compared to target RNA sequenceand/or antisense strand sequence as compared to sense strand sequence,with preferred location(s) for placement of such in/del nucleotidescorresponding to those locations described above for positioning ofmismatched and/or mismatch-forming base pairs.

In certain embodiments, the “D” residues of any of the above structuresinclude at least one PS-DNA or PS-RNA. Optionally, the “D” residues ofany of the above structures include at least one modified nucleotidethat inhibits Dicer cleavage.

While the above-described “DNA-extended” DsiRNA agents can becategorized as either “left extended” or “right extended”, DsiRNA agentscomprising both left- and right-extended DNA-containing sequences withina single agent (e.g., both flanks surrounding a core dsRNA structure aredsDNA extensions) can also be generated and used in similar manner tothose described herein for “right-extended” and “left-extended” agents.

In some embodiments, the DsiRNA of the instant invention furthercomprises a linking moiety or domain that joins the sense and antisensestrands of a DNA:DNA-extended DsiRNA agent. Optionally, such a linkingmoiety domain joins the 3′ end of the sense strand and the 5′ end of theantisense strand. The linking moiety may be a chemical (non-nucleotide)linker, such as an oligomethylenediol linker, oligoethylene glycollinker, or other art-recognized linker moiety. Alternatively, the linkercan be a nucleotide linker, optionally including an extended loop and/ortetraloop.

In one embodiment, the DsiRNA agent has an asymmetric structure, withthe sense strand having a 27-base pair length, the antisense strandhaving a 29-nucleotide length with a 2 base 3′-overhang (and, therefore,the DsiRNA agent possesses a blunt end at the 3′ end of the sensestrand/5′ end of the antisense strand), and with deoxyribonucleotideslocated at positions 24 and 25 of the sense strand (numbering fromposition 1 at the 5′ of the sense strand) and each base paired with acognate deoxyribonucleotide of the antisense strand. In anotherembodiment, this DsiRNA agent has an asymmetric structure furthercontaining 2 deoxyribonucleotides at the 3′ end of the sense strand.

In another embodiment, the DsiRNA agent has an asymmetric structure,with the sense strand having a 30-nucleotide length, the antisensestrand having a 28-nucleotide length, with a 2 nucleotide 3′ overhangpositioned at the 3′ end of the sense strand. The 3′ end of theantisense strand and 5′ end of the sense strand of this DsiRNA agentform a blunt end, and starting from position 1 at the 5′ terminus of thesense strand, positions 1-5 are deoxyribonucleotides that hybridize toform a duplex with cognate deoxyribonucleotides of the 3′ end region ofthe antisense strand. Optionally, starting from position 1 at the 5′ endof the antisense strand, positions 11-21 of the antisense strand (incertain embodiments, positions 13-21) harbor one or more nucleotidesthat either form a mismatch base pairing with the correspondingnucleotide of the sense strand, or with the corresponding nucleotide ofthe target RNA sequence when the antisense strand and the target RNAsequence hybridize to form a duplex, or with both sense strand andtarget RNA sequence. Optionally, the ultimate and penultimatenucleotides of the 5′ terminus of the sense strand and the ultimate andpenultimate nucleotides of the 3′ end of the antisense strand compriseone or more phosphorothioates (optionally, the two antisense stranddeoxyribonucleotides, the two sense strand deoxyribonucleotides, or all4 deoxyribonucleotides constituting the ultimate and penultimateresidues of both the 5′ end of the sense strand and the 3′ end of theantisense strand possess phosphorothioates).

Modification of DsiRNAs

One major factor that inhibits the effect of double stranded RNAs(“dsRNAs”) is the degradation of dsRNAs (e.g., siRNAs and DsiRNAs) bynucleases. A 3′-exonuclease is the primary nuclease activity present inserum and modification of the 3′-ends of antisense DNA oligonucleotidesis crucial to prevent degradation (Eder et al., 1991). An RNase-T familynuclease has been identified called ERI-1 which has 3′ to 5′ exonucleaseactivity that is involved in regulation and degradation of siRNAs(Kennedy et al., 2004; Hong et al., 2005). This gene is also known asThex1 (NM_(—)02067) in mice or THEX1 (NM_(—)153332) in humans and isinvolved in degradation of histone mRNA; it also mediates degradation of3′-overhangs in siRNAs, but does not degrade duplex RNA (Yang et al.,2006). It is therefore reasonable to expect that 3′-end-stabilization ofdsRNAs, including the DsiRNAs of the instant invention, will improvestability.

XRN1 (NM_(—)019001) is a 5′ to 3′ exonuclease that resides in P-bodiesand has been implicated in degradation of mRNA targeted by miRNA(Rehwinkel et al., 2005) and may also be responsible for completingdegradation initiated by internal cleavage as directed by a siRNA. XRN2(NM_(—)012255) is a distinct 5′ to 3′ exonuclease that is involved innuclear RNA processing. Although not currently implicated in degradationor processing of siRNAs and miRNAs, these both are known nucleases thatcan degrade RNAs and may also be important to consider.

RNase A is a major endonuclease activity in mammals that degrades RNAs.It is specific for ssRNA and cleaves at the 3′-end of pyrimidine bases.SiRNA degradation products consistent with RNase A cleavage can bedetected by mass spectrometry after incubation in serum (Turner et al.,2007). The 3′-overhangs enhance the susceptibility of siRNAs to RNasedegradation. Depletion of RNase A from serum reduces degradation ofsiRNAs; this degradation does show some sequence preference and is worsefor sequences having poly A/U sequence on the ends (Haupenthal et al.,2006). This suggests the possibility that lower stability regions of theduplex may “breathe” and offer transient single-stranded speciesavailable for degradation by RNase A. RNase A inhibitors can be added toserum and improve siRNA longevity and potency (Haupenthal et al., 2007).

In 21mers, phosphorothioate or boranophosphate modifications directlystabilize the internucleoside phosphate linkage. Boranophosphatemodified RNAs are highly nuclease resistant, potent as silencing agents,and are relatively non-toxic. Boranophosphate modified RNAs cannot bemanufactured using standard chemical synthesis methods and instead aremade by in vitro transcription (IVT) (Hall et al., 2004 and Hall et al.,2006). Phosphorothioate (PS) modifications can be readily placed in anRNA duplex at any desired position and can be made using standardchemical synthesis methods, though the ability to use such modificationswithin an RNA duplex that retains RNA silencing activity can be limited.As shown herein in FIG. 2 (duplex #8), inclusion of a multiplePS-modified deoxyribonucleotide residues in a tandem seriesconfiguration that base paired with a cognate tandem series ofPS-modified deoxyribonucleotide residues abolished RNA silencingactivity of an agent that was otherwise active with only unmodifieddeoxyribonucleotides present at these residues. Because PS moieties arelikely to require greater spacing when included within an RNAduplex-containing agent in order to retain RNA inhibitory activity,extended DsiRNAs such as those described herein can provide a means ofincluding more PS modifications (either PS-DNA or PS-RNA) within asingle DsiRNA agent than would otherwise be available were no suchextension used. It is noted, however, that the PS modification showsdose-dependent toxicity, so most investigators have recommended limitedincorporation in siRNAs, historically favoring the 3′-ends whereprotection from nucleases is most important (Harborth et al., 2003; Chiuand Rana, 2003; Braasch et al., 2003; Amarzguioui et al., 2003). Moreextensive PS modification can be compatible with potent RNAi activity;however, use of sugar modifications (such as 2′-O-methyl RNA) may besuperior (Choung et al., 2006).

A variety of substitutions can be placed at the 2′-position of theribose which generally increases duplex stability (T_(m)) and cangreatly improve nuclease resistance. 2′-O-methyl RNA is a naturallyoccurring modification found in mammalian ribosomal RNAs and transferRNAs. 2′-O-methyl modification in siRNAs is known, but the preciseposition of modified bases within the duplex is important to retainpotency and complete substitution of 2′-O-methyl RNA for RNA willinactivate the siRNA. For example, a pattern that employs alternating2′-O-methyl bases can have potency equivalent to unmodified RNA and isquite stable in serum (Choung et al., 2006; Czauderna et al., 2003).

The 2′-fluoro (2′-F) modification is also compatible with dsRNA (e.g.,siRNA and DsiRNA) function; it is most commonly placed at pyrimidinesites (due to reagent cost and availability) and can be combined with2′-O-methyl modification at purine positions; 2′-F purines are availableand can also be used. Heavily modified duplexes of this kind can bepotent triggers of RNAi in vitro (Allerson et al., 2005; Prakash et al.,2005; Kraynack and Baker, 2006) and can improve performance and extendduration of action when used in vivo (Morrissey et al., 2005a; Morrisseyet al., 2005b). A highly potent, nuclease stable, blunt 19mer duplexcontaining alternative 2′-F and 2′-O-Me bases is taught by Allerson. Inthis design, alternating 2′-O-Me residues are positioned in an identicalpattern to that employed by Czauderna, however the remaining RNAresidues are converted to 2′-F modified bases. A highly potent, nucleaseresistant siRNA employed by Morrissey employed a highly potent, nucleaseresistant siRNA in vivo. In addition to 2′-O-Me RNA and 2′-F RNA, thisduplex includes DNA, RNA, inverted abasic residues, and a 3′-terminal PSinternucleoside linkage. While extensive modification has certainbenefits, more limited modification of the duplex can also improve invivo performance and is both simpler and less costly to manufacture.Soutschek et al. (2004) employed a duplex in vivo and was mostly RNAwith two 2′-O-Me RNA bases and limited 3′-terminal PS internucleosidelinkages.

Locked nucleic acids (LNAs) are a different class of 2′-modificationthat can be used to stabilize dsRNA (e.g., siRNA and DsiRNA). Patternsof LNA incorporation that retain potency are more restricted than2′-O-methyl or 2′-F bases, so limited modification is preferred (Braaschet al., 2003; Grunweller et al., 2003; Elmen et al., 2005). Even withlimited incorporation, the use of LNA modifications can improve dsRNAperformance in vivo and may also alter or improve off target effectprofiles (Mook et al., 2007).

Synthetic nucleic acids introduced into cells or live animals can berecognized as “foreign” and trigger an immune response. Immunestimulation constitutes a major class of off-target effects which candramatically change experimental results and even lead to cell death.The innate immune system includes a collection of receptor moleculesthat specifically interact with DNA and RNA that mediate theseresponses, some of which are located in the cytoplasm and some of whichreside in endosomes (Marques and Williams, 2005; Schlee et al., 2006).Delivery of siRNAs by cationic lipids or liposomes exposes the siRNA toboth cytoplasmic and endosomal compartments, maximizing the risk fortriggering a type 1 interferon (IFN) response both in vitro and in vivo(Morrissey et al., 2005b; Sioud and Sorensen, 2003; Sioud, 2005; Ma etal., 2005). RNAs transcribed within the cell are less immunogenic(Robbins et al., 2006) and synthetic RNAs that are immunogenic whendelivered using lipid-based methods can evade immune stimulation whenintroduced unto cells by mechanical means, even in vivo (Heidel et al.,2004). However, lipid based delivery methods are convenient, effective,and widely used. Some general strategy to prevent immune responses isneeded, especially for in vivo application where all cell types arepresent and the risk of generating an immune response is highest. Use ofchemically modified RNAs may solve most or even all of these problems.

Although certain sequence motifs are clearly more immunogenic thanothers, it appears that the receptors of the innate immune system ingeneral distinguish the presence or absence of certain basemodifications which are more commonly found in mammalian RNAs than inprokaryotic RNAs. For example, pseudouridine, N6-methyl-A, and2′-O-methyl modified bases are recognized as “self” and inclusion ofthese residues in a synthetic RNA can help evade immune detection(Kariko et al., 2005). Extensive 2′-modification of a sequence that isstrongly immunostimulatory as unmodified RNA can block an immuneresponse when administered to mice intravenously (Morrissey et al.,2005b). However, extensive modification is not needed to escape immunedetection and substitution of as few as two 2′-O-methyl bases in asingle strand of a siRNA duplex can be sufficient to block a type 1 IFNresponse both in vitro and in vivo; modified U and G bases are mosteffective (Judge et al., 2006). As an added benefit, selectiveincorporation of 2′-O-methyl bases can reduce the magnitude ofoff-target effects (Jackson et al., 2006). Use of 2′-O-methyl basesshould therefore be considered for all dsRNAs intended for in vivoapplications as a means of blocking immune responses and has the addedbenefit of improving nuclease stability and reducing the likelihood ofoff-target effects.

Although cell death can result from immune stimulation, assessing cellviability is not an adequate method to monitor induction of IFNresponses. IFN responses can be present without cell death, and celldeath can result from target knockdown in the absence of IFN triggering(for example, if the targeted gene is essential for cell viability).Relevant cytokines can be directly measured in culture medium and avariety of commercial kits exist which make performing such assaysroutine. While a large number of different immune effector molecules canbe measured, testing levels of IFN-α, TNF-α, and IL-6 at 4 and 24 hourspost transfection is usually sufficient for screening purposes. It isimportant to include a “transfection reagent only control” as cationiclipids can trigger immune responses in certain cells in the absence ofany nucleic acid cargo. Including controls for IFN pathway inductionshould be considered for cell culture work. It is essential to test forimmune stimulation whenever administering nucleic acids in vivo, wherethe risk of triggering IFN responses is highest.

Modifications can be included in the DsiRNA agents of the presentinvention so long as the modification does not prevent the DsiRNA agentfrom serving as a substrate for Dicer. Indeed, one surprising finding ofthe instant invention is that base paired deoxyribonucleotides can beattached to previously described DsiRNA molecules, resulting in enhancedRNAi efficacy and duration, provided that such extension is performed ina region of the extended molecule that does not interfere with Dicerprocessing (e.g., 3′ of the Dicer cleavage site of the sense strand/5′of the Dicer cleavage site of the antisense strand). In one embodiment,one or more modifications are made that enhance Dicer processing of theDsiRNA agent. In a second embodiment, one or more modifications are madethat result in more effective RNAi generation. In a third embodiment,one or more modifications are made that support a greater RNAi effect.In a fourth embodiment, one or more modifications are made that resultin greater potency per each DsiRNA agent molecule to be delivered to thecell. Modifications can be incorporated in the 3′-terminal region, the5′-terminal region, in both the 3′-terminal and 5′-terminal region or insome instances in various positions within the sequence. With therestrictions noted above in mind, any number and combination ofmodifications can be incorporated into the DsiRNA agent. Where multiplemodifications are present, they may be the same or different.Modifications to bases, sugar moieties, the phosphate backbone, andtheir combinations are contemplated. Either 5′-terminus can bephosphorylated.

Examples of modifications contemplated for the phosphate backboneinclude phosphonates, including methylphosphonate, phosphorothioate, andphosphotriester modifications such as alkylphosphotriesters, and thelike. Examples of modifications contemplated for the sugar moietyinclude 2′-alkyl pyrimidine, such as 2′-O-methyl, 2′-fluoro, amino, anddeoxy modifications and the like (see, e.g., Amarzguioui et al., 2003).Examples of modifications contemplated for the base groups includeabasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil,5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil and the like.Locked nucleic acids, or LNA's, could also be incorporated. Many othermodifications are known and can be used so long as the above criteriaare satisfied. Examples of modifications are also disclosed in U.S. Pat.Nos. 5,684,143, 5,858,988 and 6,291,438 and in U.S. published patentapplication No. 2004/0203145 A1. Other modifications are disclosed inHerdewijn (2000), Eckstein (2000), Rusckowski et al. (2000), Stein etal. (2001); Vorobjev et al. (2001).

One or more modifications contemplated can be incorporated into eitherstrand. The placement of the modifications in the DsiRNA agent cangreatly affect the characteristics of the DsiRNA agent, includingconferring greater potency and stability, reducing toxicity, enhanceDicer processing, and minimizing an immune response. In one embodiment,the antisense strand or the sense strand or both strands have one ormore 2′-O-methyl modified nucleotides. In another embodiment, theantisense strand contains 2′-O-methyl modified nucleotides. In anotherembodiment, the antisense stand contains a 3′ overhang that is comprisedof 2′-O-methyl modified nucleotides. The antisense strand could alsoinclude additional 2′-O-methyl modified nucleotides.

In certain embodiments of the present invention, the DsiRNA agent hasone or more properties which enhance its processing by Dicer. Accordingto these embodiments, the DsiRNA agent has a length sufficient such thatit is processed by Dicer to produce an active siRNA and at least one ofthe following properties: (i) the DsiRNA agent is asymmetric, e.g., hasa 3′ overhang on the antisense strand and (ii) the DsiRNA agent has amodified 3′ end on the sense strand to direct orientation of Dicerbinding and processing of the dsRNA region to an active siRNA. Incertain such embodiments, the presence of one or more base paireddeoxyribonucleotides in a region of the sense strand that is 3′ to theprojected site of Dicer enzyme cleavage and corresponding region of theantisense strand that is 5′ of the projected site of Dicer enzymecleavage can also serve to orient such a molecule for appropriatedirectionality of Dicer enzyme cleavage.

In certain embodiments, the length of the dsDNA region (or length of theregion comprising DNA:DNA base pairs) is 1-50 base pairs, optionally2-30 base pairs, preferably 2-20 base pairs, and more preferably 2-15base pairs. Thus, a DNA:DNA-extended DsiRNA of the instant invention maypossess a dsDNA region that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50 or more (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more)base pairs in length.

In some embodiments, the longest strand in the dsNA comprises 29-43nucleotides. In one embodiment, the DsiRNA agent is asymmetric such thatthe 3′ end of the sense strand and 5′ end of the antisense strand form ablunt end, and the 3′ end of the antisense strand overhangs the 5′ endof the sense strand. In certain embodiments, the 3′ overhang of theantisense strand is 1-10 nucleotides, and optionally is 1-4 nucleotides,for example 2 nucleotides. Both the sense and the antisense strand mayalso have a 5′ phosphate.

In certain embodiments, the sense strand of a DsiRNA of the inventionthat comprises base paired deoxyribonucleotide residues has a totallength of between 26 nucleotides and 39 or more nucleotides (e.g., thesense strand possesses a length of 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more(e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more) nucleotides). Incertain embodiments, the length of the sense strand is between 26nucleotides and 39 nucleotides, optionally between 27 and 35nucleotides, or, optionally, between 27 and 33 nucleotides in length. Inrelated embodiments, the antisense strand has a length of between 27 and43 or more nucleotides (e.g., the sense strand possesses a length of 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50 or more (e.g., 51, 52, 53, 54, 55, 56, 57, 58,59, 60 or more) nucleotides). In certain such embodiments, the antisensestrand has a length of between 27 and 43 nucleotides in length, orbetween 27 and 39 nucleotides in length, or between 27 and 35nucleotides in length, or between 28 and 37 nucleotides in length, or,optionally, between 29 and 35 nucleotides in length.

In certain embodiments, the presence of one or more base paireddeoxyribonucleotides in a region of the sense strand that is 3′ of theprojected site of Dicer enzyme cleavage and corresponding region of theantisense strand that is 5′ of the projected site of Dicer enzymecleavage can serve to direct Dicer enzyme cleavage of such a molecule.While certain exemplified agents of the invention possess a sense stranddeoxyribonucleotide that is located at position 24 or more 3′ whencounting from position 1 at the 5′ end of the sense strand, and havingthis position 24 or more 3′ deoxyribonucleotide of the sense strand basepairing with a cognate deoxyribonucleotide of the antisense strand, insome embodiments, it is also possible to direct Dicer to cleave ashorter product, e.g., a 19mer or a 20mer via inclusion ofdeoxyribonucleotide residues at, e.g., position 20 of the sense strand.Such a position 20 deoxyribonucleotide base pairs with a correspondingdeoxyribonucleotide of the antisense strand, thereby directingDicer-mediated excision of a 19mer as the most prevalent Dicer product(it is noted that the antisense strand can also comprise one or twodeoxyribonucleotide residues immediately 3′ of the antisense residuethat base pairs with the position 20 deoxyribonucleotide residue of thesense strand in such embodiments, to further direct Dicer cleavage ofthe antisense strand). In such embodiments, the double-stranded DNAregion (which is inclusive of modified nucleic acids that block Dicercleavage) will generally possess a length of greater than 1 or 2 basepairs (e.g., 3 to 5 base pairs or more), in order to direct Dicercleavage to generate what is normally a non-preferred length of Dicercleavage product. A parallel approach can also be taken to direct Dicerexcision of 20mer siRNAs, with the positioning of the firstdeoxyribonucleotide residue of the sense strand (when surveying thesense strand from position 1 at the 5′ terminus of the sense strand)occurring at position 21.

In certain embodiments, the sense strand of the DsiRNA agent is modifiedfor Dicer processing by suitable modifiers located at the 3′ end of thesense strand, i.e., the DsiRNA agent is designed to direct orientationof Dicer binding and processing via sense strand modification. Suitablemodifiers include nucleotides such as deoxyribonucleotides,dideoxyribonucleotides, acyclonucleotides and the like and stericallyhindered molecules, such as fluorescent molecules and the like.Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the2′-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotidemodifiers could include 3′-deoxyadenosine (cordycepin),3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI),2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxyribonucleotides are used as the modifiers. When nucleotidemodifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotidemodifiers are substituted for the ribonucleotides on the 3′ end of thesense strand. When sterically hindered molecules are utilized, they areattached to the ribonucleotide at the 3′ end of the antisense strand.Thus, the length of the strand does not change with the incorporation ofthe modifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the DsiRNA agent to direct the orientationof Dicer processing of the antisense strand. In a further embodiment ofthe present invention, two terminal DNA bases are substituted for tworibonucleotides on the 3′-end of the sense strand forming a blunt end ofthe duplex on the 3′ end of the sense strand and the 5′ end of theantisense strand, and a two-nucleotide RNA overhang is located on the3′-end of the antisense strand. This is an asymmetric composition withDNA on the blunt end and RNA bases on the overhanging end. In certainembodiments of the instant invention, the modified nucleotides (e.g.,deoxyribonucleotides) of the penultimate and ultimate positions of the3′ terminus of the sense strand base pair with corresponding modifiednucleotides (e.g., deoxyribonucleotides) of the antisense strand(optionally, the penultimate and ultimate residues of the 5′ end of theantisense strand in those DsiRNA agents of the instant inventionpossessing a blunt end at the 3′ terminus of the sense strand/5′terminus of the antisense strand).

The sense and antisense strands of a DsiRNA agent of the instantinvention anneal under biological conditions, such as the conditionsfound in the cytoplasm of a cell. In addition, a region of one of thesequences, particularly of the antisense strand, of the DsiRNA agent hasa sequence length of at least 19 nucleotides, wherein these nucleotidesare in the 21-nucleotide region adjacent to the 3′ end of the antisensestrand and are sufficiently complementary to a nucleotide sequence ofthe RNA produced from the target gene to anneal with and/or decreaselevels of such a target RNA.

The DsiRNA agent of the instant invention may possess one or moredeoxyribonucleotide base pairs located at any positions of sense andantisense strands that are located 3′ of the projected Dicer cleavagesite of the sense strand and 5′ of the projected Dicer cleavage site ofthe antisense strand. In certain embodiments, one, two, three or allfour of positions 24-27 of the sense strand (starting from position 1 atthe 5′ terminus of the sense strand) are deoxyribonucleotides, eachdeoxyribonucleotide of which base pairs with a correspondingdeoxyribonucleotide of the antisense strand. In certain embodiments, thedeoxyribonucleotides of the 5′ region of the antisense strand (e.g., theregion of the antisense strand located 5′ of the projected Dicercleavage site for a given DsiRNA molecule) are not complementary to thetarget RNA to which the DsiRNA agent is directed. In relatedembodiments, the entire region of the antisense strand located 5′ of theprojected Dicer cleavage site of a DsiRNA agent is not complementary tothe target RNA to which the DsiRNA agent is directed. In certainembodiments, the deoxyribonucleotides of the antisense strand or theentire region of the antisense strand that is located 5′ of theprojected Dicer cleavage site of the DsiRNA agent is not sufficientlycomplementary to the target RNA to enhance annealing of the antisensestrand of the DsiRNA to the target RNA when the antisense strand isannealed to the target RNA under conditions sufficient to allow forannealing between the antisense strand and the target RNA (e.g., a“core” antisense strand sequence lacking the DNA-extended region annealsequally well to the target RNA as the same “core” antisense strandsequence also extended with sequence of the DNA-extended region).

The DsiRNA agent may also have one or more of the following additionalproperties: (a) the antisense strand has a right or left shift from thetypical 21mer, (b) the strands may not be completely complementary,i.e., the strands may contain simple mismatch pairings and (c) basemodifications such as locked nucleic acid(s) may be included in the 5′end of the sense strand. A “typical” 21mer siRNA is designed usingconventional techniques. In one technique, a variety of sites arecommonly tested in parallel or pools containing several distinct siRNAduplexes specific to the same target with the hope that one of thereagents will be effective (Ji et al., 2003). Other techniques usedesign rules and algorithms to increase the likelihood of obtainingactive RNAi effector molecules (Schwarz et al., 2003; Khvorova et al.,2003; Ui-Tei et al., 2004; Reynolds et al., 2004; Krol et al., 2004;Yuan et al., 2004; Boese et al., 2005). High throughput selection ofsiRNA has also been developed (U.S. published patent application No.2005/0042641 A1). Potential target sites can also be analyzed bysecondary structure predictions (Heale et al., 2005). This 21mer is thenused to design a right shift to include 3-9 additional nucleotides onthe 5′ end of the 21mer. The sequence of these additional nucleotidesmay have any sequence. In one embodiment, the added ribonucleotides arebased on the sequence of the target gene. Even in this embodiment, fullcomplementarity between the target sequence and the antisense siRNA isnot required.

The first and second oligonucleotides of a DsiRNA agent of the instantinvention are not required to be completely complementary. They onlyneed to be substantially complementary to anneal under biologicalconditions and to provide a substrate for Dicer that produces a siRNAsufficiently complementary to the target sequence. Locked nucleic acids,or LNA's, are well known to a skilled artisan (Elman et al., 2005;Kurreck et al., 2002; Crinelli et al., 2002; Braasch and Corey, 2001;Bondensgaard et al., 2000; Wahlestedt et al., 2000). In one embodiment,an LNA is incorporated at the 5′ terminus of the sense strand. Inanother embodiment, an LNA is incorporated at the 5′ terminus of thesense strand in duplexes designed to include a 3′ overhang on theantisense strand.

In certain embodiments, the DsiRNA agent of the instant invention has anasymmetric structure, with the sense strand having a 27-base pairlength, and the antisense strand having a 29-base pair length with a 2base 3′-overhang. Such agents optionally may possess between one andfour deoxyribonucleotides of the 3′ terminal region (specifically, theregion 3′ of the projected Dicer cleavage site) of the sense strand, atleast one of which base pairs with a cognate deoxyribonucleotide of the5′ terminal region (specifically, the region 5′ of the projected Dicercleavage site) of the antisense strand. In other embodiments, the sensestrand has a 28-base pair length, and the antisense strand has a 30-basepair length with a 2 base 3′-overhang. Such agents optionally maypossess between one and five deoxyribonucleotides of the 3′ terminalregion (specifically, the region 3′ of the projected Dicer cleavagesite) of the sense strand, at least one of which base pairs with acognate deoxyribonucleotide of the 5′ terminal region (specifically, theregion 5′ of the projected Dicer cleavage site) of the antisense strand.In additional embodiments, the sense strand has a 29-base pair length,and the antisense strand has a 31-base pair length with a 2 base3′-overhang. Such agents optionally possess between one and sixdeoxyribonucleotides of the 3′ terminal region (specifically, the region3′ of the projected Dicer cleavage site) of the sense strand, at leastone of which base pairs with a cognate deoxyribonucleotide of the 5′terminal region (specifically, the region 5′ of the projected Dicercleavage site) of the antisense strand. In further embodiments, thesense strand has a 30-base pair length, and the antisense strand has a32-base pair length with a 2 base 3′-overhang. Such agents optionallypossess between one and seven deoxyribonucleotides of the 3′ terminalregion (specifically, the region 3′ of the projected Dicer cleavagesite) of the sense strand, at least one of which base pairs with acognate deoxyribonucleotide of the 5′ terminal region (specifically, theregion 5′ of the projected Dicer cleavage site) of the antisense strand.In other embodiments, the sense strand has a 31-base pair length, andthe antisense strand has a 33-base pair length with a 2 base3′-overhang. Such agents optionally possess between one and eightdeoxyribonucleotides of the 3′ terminal region (specifically, the region3′ of the projected Dicer cleavage site) of the sense strand, at leastone of which base pairs with a cognate deoxyribonucleotide of the 5′terminal region (specifically, the region 5′ of the projected Dicercleavage site) of the antisense strand. In additional embodiments, thesense strand has a 32-base pair length, and the antisense strand has a34-base pair length with a 2 base 3′-overhang. Such agents optionallypossess between one and nine deoxyribonucleotides of the 3′ terminalregion (specifically, the region 3′ of the projected Dicer cleavagesite) of the sense strand, at least one of which base pairs with acognate deoxyribonucleotide of the 5′ terminal region (specifically, theregion 5′ of the projected Dicer cleavage site) of the antisense strand.In certain further embodiments, the sense strand has a 33-base pairlength, and the antisense strand has a 35-base pair length with a 2 base3′-overhang. Such agents optionally possess between one and tendeoxyribonucleotides of the 3′ terminal region (specifically, the region3′ of the projected Dicer cleavage site) of the sense strand, at leastone of which base pairs with a cognate deoxyribonucleotide of the 5′terminal region (specifically, the region 5′ of the projected Dicercleavage site) of the antisense strand. In still other embodiments, anyof these DsiRNA agents have an asymmetric structure that furthercontains 2 deoxyribonucleotides at the 3′ end of the sense strand inplace of two of the ribonucleotides; optionally, these 2deoxyribonucleotides base pair with cognate deoxyribonucleotides of theantisense strand.

Certain DsiRNA agent compositions containing two separateoligonucleotides can be linked by a third structure. The third structurewill not block Dicer activity on the DsiRNA agent and will not interferewith the directed destruction of the RNA transcribed from the targetgene. In one embodiment, the third structure may be a chemical linkinggroup. Many suitable chemical linking groups are known in the art andcan be used. Alternatively, the third structure may be anoligonucleotide that links the two oligonucleotides of the DsiRNA agentin a manner such that a hairpin structure is produced upon annealing ofthe two oligonucleotides making up the dsNA composition. The hairpinstructure will not block Dicer activity on the DsiRNA agent and will notinterfere with the directed destruction of the target RNA.

In certain embodiments, the DsiRNA agent of the invention has severalproperties which enhance its processing by Dicer. According to suchembodiments, the DsiRNA agent has a length sufficient such that it isprocessed by Dicer to produce an siRNA and at least one of the followingproperties: (i) the DsiRNA agent is asymmetric, e.g., has a 3′ overhangon the sense strand and (ii) the DsiRNA agent has a modified 3′ end onthe antisense strand to direct orientation of Dicer binding andprocessing of the dsRNA region to an active siRNA. According to theseembodiments, the longest strand in the DsiRNA agent comprises 25-43nucleotides. In one embodiment, the sense strand comprises 25-39nucleotides and the antisense strand comprises 26-43 nucleotides. Theresulting dsNA can have an overhang on the 3′ end of the sense strand.The overhang is 1-4 nucleotides, such as 2 nucleotides. The antisense orsense strand may also have a 5′ phosphate.

In certain embodiments, the sense strand of a DsiRNA agent is modifiedfor Dicer processing by suitable modifiers located at the 3′ end of thesense strand, i.e., the DsiRNA agent is designed to direct orientationof Dicer binding and processing. Suitable modifiers include nucleotidessuch as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotidesand the like and sterically hindered molecules, such as fluorescentmolecules and the like. Acyclonucleotides substitute a2-hydroxyethoxymethyl group for the 2′-deoxyribofuranosyl sugar normallypresent in dNMPs. Other nucleotide modifiers could include3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxyribonucleotides are used as the modifiers. When nucleotidemodifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotidemodifiers are substituted for the ribonucleotides on the 3′ end of thesense strand. When sterically hindered molecules are utilized, they areattached to the ribonucleotide at the 3′ end of the antisense strand.Thus, the length of the strand does not change with the incorporation ofthe modifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the dsNA to direct the orientation ofDicer processing. In a further embodiment, two terminal DNA bases arelocated on the 3′ end of the sense strand in place of tworibonucleotides forming a blunt end of the duplex on the 5′ end of theantisense strand and the 3′ end of the sense strand, and atwo-nucleotide RNA overhang is located on the 3′-end of the antisensestrand. This is an asymmetric composition with DNA on the blunt end andRNA bases on the overhanging end.

In certain other embodiments, the antisense strand of a DsiRNA agent ismodified for Dicer processing by suitable modifiers located at the 3′end of the antisense strand, i.e., the DsiRNA agent is designed todirect orientation of Dicer binding and processing. Suitable modifiersinclude nucleotides such as deoxyribonucleotides,dideoxyribonucleotides, acyclonucleotides and the like and stericallyhindered molecules, such as fluorescent molecules and the like.Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the2′-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotidemodifiers could include 3′-deoxyadenosine (cordycepin),3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI),2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxyribonucleotides are used as the modifiers. When nucleotidemodifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotidemodifiers are substituted for the ribonucleotides on the 3′ end of theantisense strand. When sterically hindered molecules are utilized, theyare attached to the ribonucleotide at the 3′ end of the antisensestrand. Thus, the length of the strand does not change with theincorporation of the modifiers. In another embodiment, the inventioncontemplates substituting two DNA bases in the dsNA to direct theorientation of Dicer processing. In a further invention, two terminalDNA bases are located on the 3′ end of the antisense strand in place oftwo ribonucleotides forming a blunt end of the duplex on the 5′ end ofthe sense strand and the 3′ end of the antisense strand, and atwo-nucleotide RNA overhang is located on the 3′-end of the sensestrand. This is also an asymmetric composition with DNA on the blunt endand RNA bases on the overhanging end.

The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsNA has a sequence length of at least 19 nucleotides, wherein thesenucleotides are adjacent to the 3′ end of antisense strand and aresufficiently complementary to a nucleotide sequence of the target RNA todirect RNA interference.

Additionally, the DsiRNA agent structure can be optimized to ensure thatthe oligonucleotide segment generated from Dicer's cleavage will be theportion of the oligonucleotide that is most effective in inhibiting geneexpression. For example, in one embodiment of the invention, a 27-35-bpoligonucleotide of the DsiRNA agent structure is synthesized wherein theanticipated 21 to 22-bp segment that will inhibit gene expression islocated on the 3′-end of the antisense strand. The remaining baseslocated on the 5′-end of the antisense strand will be cleaved by Dicerand will be discarded. This cleaved portion can be homologous (i.e.,based on the sequence of the target sequence) or non-homologous andadded to extend the nucleic acid strand. As surprisingly identified inthe instant invention, such extension can be performed with base pairedDNA residues (double stranded DNA:DNA extensions), resulting in extendedDsiRNA agents having improved efficacy or duration of effect thancorresponding double stranded RNA:RNA-extended DsiRNA agents.

US 2007/0265220 discloses that 27mer DsiRNAs show improved stability inserum over comparable 21mer siRNA compositions, even absent chemicalmodification. Modifications of DsiRNA agents, such as inclusion of2′-O-methyl RNA in the antisense strand, in patterns such as detailed inUS 2007/0265220 and in the instant Examples, when coupled with additionof a 5′ Phosphate, can improve stability of DsiRNA agents. Addition of5′-phosphate to all strands in synthetic RNA duplexes may be aninexpensive and physiological method to confer some limited degree ofnuclease stability.

The chemical modification patterns of the DsiRNA agents of the instantinvention are designed to enhance the efficacy of such agents.Accordingly, such modifications are designed to avoid reducing potencyof DsiRNA agents; to avoid interfering with Dicer processing of DsiRNAagents; to improve stability in biological fluids (reduce nucleasesensitivity) of DsiRNA agents; or to block or evade detection by theinnate immune system. Such modifications are also designed to avoidbeing toxic and to avoid increasing the cost or impact the ease ofmanufacturing the instant DsiRNA agents of the invention.

RNA Processing

siRNA

The process of siRNA-mediated RNAi is triggered by the presence of long,dsRNA molecules in a cell. During the initiation step of RNAi, thesedsRNA molecules are cleaved into 21-23 nucleotide (nt) small-interferingRNA duplexes (siRNAs) by Dicer, a conserved family of enzymes containingtwo RNase III-like domains (Bernstein et al. 2001; Elbashir et al.2001). The siRNAs are characterized by a 19-21 base pair duplex regionand 2 nucleotide 3′ overhangs on each strand. During the effector stepof RNAi, the siRNAs become incorporated into a multimeric proteincomplex called RNA-induced silencing complex (RISC), where they serve asguides to select fully complementary mRNA substrates for degradation.Degradation is initiated by endonucleolytic cleavage of the mRNA withinthe region complementary to the siRNA. More precisely, the mRNA iscleaved at a position 10 nucleotides from the 5′ end of the guidingsiRNA (Elbashir et al. 2001 Genes & Dev. 15: 188-200; Nykanen et al.2001 Cell 107: 309-321; Martinez et al. 2002 Cell 110: 563-574). Anendonuclease responsible for this cleavage was identified as Argonaute2(Ago2; Liu et al. Science, 305: 1437-41).

miRNA

The majority of human miRNAs (70%)—and presumably the majority of miRNAsof other mammals—are transcribed from introns and/or exons, andapproximately 30% are located in intergenic regions (Rodriguez et al.,Genome Res. 2004, 14(10A), 1902-1910). In human and animal, miRNAs areusually transcribed by RNA polymerase II (Farh et al. Science 2005,310(5755), 1817-1821), and in some cases by pol III (Borchert et al.Nat. Struct. Mol. Biol. 2006, 13(12), 1097-1101). Certain viral encodedmiRNAs are transcribed by RNA polymerase III (Pfeffer et al. Nat.Methods 2005, 2(4), 269-276; Andersson et al. J. Virol. 2005, 79(15),9556-9565), and some are located in the open reading frame of viral gene(Pfeffer et al. Nat. Methods 2005, 2(4), 269-276; Samols et al. J.Virol. 2005, 79(14), 9301-9305). miRNA transcription results in theproduction of large monocistronic, bicistronic or polycistronic primarytranscripts (pri-miRNAs). A single pri-miRNA may range fromapproximately 200 nucleotides (nt) to several kilobases (kb) in lengthand have both a 5′ 7-methylguanosine (m7) caps and a 3′ poly (A) tail.Characteristically, the mature miRNA sequences are localized to regionsof imperfect stem-loop sequences within the pri-miRNAs (Cullen, Mol.Cell 2004, 16(6), 861-865).

The first step of miRNA maturation in the nucleus is the recognition andcleavage of the pri-miRNAs by the RNase III Drosha-DGCR8 nuclearmicroprocessor complex, which releases a ˜70 nt hairpin-containingprecursor molecule called pre-miRNAs, with a monophosphate at the 5′terminus and a 2-nt overhang with a hydroxyl group at the 3′ terminus(Cai et al. RNA 2004, 10(12), 1957-1966; Lee et al. Nature 2003,425(6956), 415-419; Kim Nat. Rev. Mol. Cell. Biol. 2005, 6(5), 376-385).The next step is the nuclear transport of the pre-miRNAs out of thenucleus into the cytoplasm by Exportin-5, a carrier protein (Yi et al.Genes. Dev. 2003, 17(24), 3011-3016, Bohnsack et al. RNA 2004, 10(2),185-191). Exportin-5 and the GTP-bound form of its cofactor Ran togetherrecognize and bind the 2 nucleotide 3′ overhang and the adjacent stemthat are characteristics of pre-miRNA (Basyuk et al. Nucl. Acids Res.2003, 31(22), 6593-6597, Zamore Mol. Cell. 2001, 8(6), 1158-1160). Inthe cytoplasm, GTP hydrolysis results in release of the pre-miRNA, whichis then processed by a cellular endonuclease III enzyme Dicer (Bohnsacket al.). Dicer was first recognized for its role in generating siRNAsthat mediate RNA interference (RNAi). Dicer acts in concert with itscofactors TRBP (Transactivating region binding protein; Chendrimata etal. Nature 2005, 436(7051), 740-744) and PACT (interferon-inducibledouble strand-RNA-dependant protein kinase activator; Lee et al. EMBO J.2006, 25(3), 522-532). These enzymes bind at the 3′ 2 nucleotideoverhang at the base of the pre-miRNA hairpin and remove the terminalloop, yielding an approximately 21-nt miRNA duplex intermediate withboth termini having 5′ monophosphates, 3′ 2 nucleotide overhangs and 3′hydroxyl groups. The miRNA guide strand, the 5′ terminus of which isenergetically less stable, is then selected for incorporation into theRISC (RNA-induced silencing complex), while the ‘passenger’ strand isreleased and degraded (Maniataki et al. Genes. Dev. 2005, 19(24),2979-2990; Hammond et al. Nature 2000, 404(6775), 293-296). Thecomposition of RISC remains incompletely defined, but a key component isa member of the Argonaute (Ago) protein family (Maniataki et al.;Meister et al. Mol. Cell. 2004, 15(2), 185-197).

The mature miRNA then directs RISC to complementary mRNA species. If thetarget mRNA has perfect complementarity to the miRNA-armed RISC, themRNA will be cleaved and degraded (Zeng et al. Proc. Natl. Acad. Sci.USA 2003, 100(17), 9779-9784; Hutvagner et al. Science 2002, 297(55 89),2056-2060). But as the most common situation in mammalian cells, themiRNAs targets mRNAs with imperfect complementarity and suppress theirtranslation, resulting in reduced expression of the correspondingproteins (Yekta et al. Science 2004, 304(5670), 594-596; Olsen et al.Dev. Biol. 1999, 216(2), 671-680). The 5′ region of the miRNA,especially the match between miRNA and target sequence at nucleotides2-7 or 8 of miRNA (starting from position 1 at the 5′ terminus), whichis called the seed region, is essentially important for miRNA targeting,and this seed match has also become a key principle widely used incomputer prediction of the miRNA targeting (Lewis et al. Cell 2005,120(1), 15-20; Brennecke et al. PLoS Biol. 2005, 3(3), e85). miRNAregulation of the miRNA-mRNA duplexes is mediated mainly throughmultiple complementary sites in the 3′ UTRs, but there are manyexceptions. miRNAs may also bind the 5′ UTR and/or the coding region ofmRNAs, resulting in a similar outcome (Lytle et al. Proc. Natl. Acad.Sci. USA 2007, 104(23), 9667-9672).

RNase H

RNase H is a ribonuclease that cleaves the 3′-O-P bond of RNA in aDNA/RNA duplex to produce 3′-hydroxyl and 5′-phosphate terminatedproducts. RNase H is a non-specific endonuclease and catalyzes cleavageof RNA via a hydrolytic mechanism, aided by an enzyme-bound divalentmetal ion. Members of the RNase H family are found in nearly allorganisms, from archaea and prokaryotes to eukaryotes. During DNAreplication, RNase H is believed to cut the RNA primers responsible forpriming generation of Okazaki fragments; however, the RNase H enzyme maybe more generally employed to cleave any DNA:RNA hybrid sequence ofsufficient length (e.g., typically DNA:RNA hybrid sequences of 4 or morebase pairs in length in mammals).

MicroRNA and MicroRNA-Like Therapeutics

MicroRNAs (miRNAs) have been described to act by binding to the 3′ UTRof a template transcript, thereby inhibiting expression of a proteinencoded by the template transcript by a mechanism related to butdistinct from classic RNA interference. Specifically, miRNAs arebelieved to act by reducing translation of the target transcript, ratherthan by decreasing its stability. Naturally-occurring miRNAs aretypically approximately 22 nt in length. It is believed that they arederived from larger precursors known as small temporal RNAs (stRNAs)approximately 70 nt long.

Interference agents such as siRNAs, and more specifically such asmiRNAs, that bind within the 3′ UTR (or elsewhere in a targettranscript, e.g., in repeated elements of, e.g., Notch and/ortranscripts of the Notch family) and inhibit translation may tolerate alarger number of mismatches in the siRNA/template (miRNA/template)duplex, and particularly may tolerate mismatches within the centralregion of the duplex. In fact, there is evidence that some mismatchesmay be desirable or required, as naturally occurring stRNAs frequentlyexhibit such mismatches, as do miRNAs that have been shown to inhibittranslation in vitro (Zeng et al., Molecular Cell, 9: 1-20). Forexample, when hybridized with the target transcript, such miRNAsfrequently include two stretches of perfect complementarity separated bya region of mismatch. Such a hybridized complex commonly includes tworegions of perfect complementarily (duplex portions) comprisingnucleotide pairs, and at least a single mismatched base pair, which maybe, e.g., G:A, G:U, G:G, A:A, A:C, U:U, U:C, C:C, G:-, A:-, U:-, C:-,etc.. Such mismatched nucleotides, especially if present in tandem(e.g., a two, three or four nucleotide area of mismatch) can form abulge that separates duplex portions which are located on either flankof such a bulge. A variety of structures are possible. For example, themiRNA may include multiple areas of nonidentity (mismatch). The areas ofnonidentity (mismatch) need not be symmetrical in the sense that boththe target and the miRNA include nonpaired nucleotides. For example,structures have been described in which only one strand includesnonpaired nucleotides (Zeng et al., FIG. 33). Typically the stretches ofperfect complementarily within a miRNA agent are at least 5 nucleotidesin length, e.g., 6, 7, or more nucleotides in length, while the regionsof mismatch may be, for example, 1, 2, 3, or 4 nucleotides in length.

In general, any particular siRNA could function to inhibit geneexpression both via (i) the “classical” siRNA pathway, in whichstability of a target transcript is reduced and in which perfectcomplementarily between the siRNA and the target is frequentlypreferred, and also by (ii) the “alternative” pathway (generallycharacterized as the miRNA pathway in animals), in which translation ofa target transcript is inhibited. Generally, the transcripts targeted bya particular siRNA via mechanism (i) would be distinct from thetranscript targeted via mechanism (ii), although it is possible that asingle transcript could contain regions that could serve as targets forboth the classical and alternative pathways. (Note that the terms“classical” and “alternative” are used merely for convenience andgenerally are believed to reflect historical timing of discovery of suchmechanisms in animal cells, but do not reflect the importance,effectiveness, or other features of either mechanism.) One common goalof siRNA design has been to target a single transcript with greatspecificity, via mechanism (i), while minimizing off-target effects,including those effects potentially elicited via mechansim (ii).However, it is among the goals of the instant invention to provide RNAinterference agents that possess mismatch residues by design, either forpurpose of mimicking the activities of naturally-occurring miRNAs, or tocreate agents directed against target RNAs for which no correspondingmiRNA is presently known, with the inhibitory and/or therapeuticefficacies/potencies of such mismatch-containing DsiRNA agents (e.g.,DsiRNAmm agents) tolerant of, and indeed possibly enhanced by, suchmismatches.

The tolerance of miRNA agents for mismatched nucleotides (and, indeedthe existence and natural use of mechanism (ii) above in the cell)suggests the use of miRNAs in manners that are advantageous to and/orexpand upon the “classical” use of perfectly complementary siRNAs thatact via mechanism (i). Because miRNAs are naturally occurring molecules,there are likely to be distinct advantages in applying miRNAs astherapeutic agents. miRNAs benefit from hundreds of millions of years ofevolutionary “fine tuning” of their function, Thus, sequence-specific“off target” effects should not be an issue with naturally occurringmiRNAs, nor, by extension, with certain synthetic DsiRNAs of theinvention (e.g., DsiRNAmm agents) designed to mimic naturally occurringmiRNAs. In addition, miRNAs have evolved to modulate the expression ofgroups of genes, driving both up and down regulation (in certaininstances, performing both functions concurrently within a cell with asingle miRNA acting promiscuously upon multiple target RNAs), with theresult that complex cell functions can be precisely modulated. Suchreplacement of naturally occurring miRNAs can involve introducingsynthetic miRNAs or miRNA mimetics (e.g., certain DsiRNAmms) intodiseased tissues in an effort to restore normal proliferation,apoptosis, cell cycle, and other cellular functions that have beenaffected by down-regulation of one or more miRNAs. in certain instances,reactivation of these miRNA-regulated pathways has produced asignificant therapeutic response (e.g., In one study on cardiachypertrophy, overexpression of miR-133 by adenovirus-mediated deliveryof a miRNA expression cassette protected animals from agonist-inducedcardiac hypertrophy, whereas reciprocally reduction of miR-133 inwild-type mice by antagomirs caused an increase in hypertrophic markers(Care et al. Nat. Med. 13: 613-618)).

To date, more than 600 miRNAs have been identified as encoded within thehuman genome, with such miRNAs expressed and processed by a combinationof proteins in the nucleus and cytoplasm. miRNAs are highly conservedamong vertebrates and comprise approximately 2% of all mammalian genes.Since each miRNA appears to regulate the expression of multiple, e.g.,two, three, four, five, six, seven, eight, nine or even tens to hundredsof different genes, miRNAs can function as “master-switches”,efficiently regulating and coordinating multiple cellular pathways andprocesses. By coordinating the expression of multiple genes, miRNAs playkey roles in embryonic development, immunity, inflammation, as well ascellular growth and proliferation.

Expression and functional studies suggest that the altered expression ofspecific miRNAs is critical to a variety of human diseases. Mountingevidence indicates that the introduction of specific miRNAs into diseasecells and tissues can induce favorable therapeutic responses (Pappas etal., Expert Opin Ther Targets. 12: 115-27) The promise of miRNA therapyis perhaps greatest in cancer due to the apparent role of certain miRNAsas tumor suppressors. The rationale for miRNA-based therapeutics for,e.g., cancer is supported, at least in part, by the followingobservations:

-   -   (1) miRNAs are frequently mis-regulated and expressed at altered        levels in diseased tissues when compared to normal tissues. A        number of studies have shown altered. levels of miRAs in        cancerous tissues relative to their corresponding normal        tissues. Often, altered expression is the consequence of genetic        mutations that lead to increased or reduced expression of        particular miRNAs. Diseases that possess unique miRNA expression        signatures can be exploited as diagnostic and prognostic        markers, and can be targeted with the DsiRNA DsiRNAmm) agents of        the invention,    -   (2) Mis-regulated miRNAs contribute to cancer development by        functioning as oncogenes or tumor suppressors. Oncogenes are        defined as genes whose over-expression or inappropriate        activation leads to oncogenesis. Tumor suppressors are genes        that are required to keep cells from being cancerous; the        down-regulation or inactivation of tumor suppressors is a common        inducer of cancer. Both types of genes represent preferred drug        targets, as such targeting can specifically act upon the        molecular basis for a particular cancer. Examples of oncogenic        miRNAs are miR-155 and miR-17-92; let-7 is an example of a tumor        suppressive miRNA.    -   (3) Administration of miRNA induces a therapeutic response by        blocking or reducing tumor growth in pre-clinical animal        studies. The scientific literature provides proof-of-concept        studies demonstrating that restoring miRNA function can prevent        or reduce the growth of cancer cells in vitro and also in animal        models. A well-characterized. example is the anti-tumor activity        of let-7 in models for breast and lung cancer. DsiRNAs (e.g.,        DsiRNAmms) of the invention which are designed to mimic let-7        can be used to target such cancers, and it is also possible to        use the DsiRNA design parameters described herein to generate        new DsiRNA (e.g., DsiRNAmm) agents directed against target RNAs        for which no counterpart naturally occurring miRNA is known        (e.g., repeats within Notch or other transcripts), to screen for        therapeutic lead compounds, e.g., agents that are capable of        reducing tumor burden in pre-clinical animal models,    -   (4) A given miRNA controls multiple cellular pathways and        therefore may have superior therapeutic activity. Based on their        biology, miRNAs can function as “master switches” of the genome,        regulating multiple gene products and coordinating multiple        pathways. Genes regulated by miRNAs include genes that encode        conventional oncogenes and tumor suppressors, many of which are        individually pursued as drug targets by the pharmaceutical        industry. Thus, miRNA therapeutics could possess activity        superior to siRNAs and other forms of lead con/pounds by        targeting multiple disease and/or cancer-associated genes.        (liven the observation that mis-regulation of miRNAs is        frequently an early event in the process of tumorigenesis, miRNA        therapeutics, which. replace missing miRNAs, may be the most        appropriate therapy.    -   (5) miRNAs are natural molecules and are, therefore less prone        to induce non-specific side-effects. Millions of years of        evolution helped to develop the regulatory network of miRNAs,        fine-tuning the interaction of miRNA with target messenger RNAs.        Therefore, miRNAs and miRNA derivatives (e.g., DsiRNAs designed        to mimic naturally occurring miRNAs) will have few if any        sequence-specific “off-target” effects when applied in the        proper context.

The physical characteristics of siRNAs and miRNAs are similar.Accordingly, technologies that are effective in delivering siRNAs (e.g.,DsiRNAs of the invention) are likewise effective in delivering syntheticmiRNAs (e.g., certain DsiRNAmms of the invention).

Conjugation and Delivery of DsiRNA Agents

In certain embodiments, the present invention relates to a method fortreating a subject having or at risk of developing a disease ordisorder. In such embodiments, the DsiRNA can act as a novel therapeuticagent for controlling the disease or disorder. The method comprisesadministering a pharmaceutical composition of the invention to thepatient (e.g., human), such that the expression, level and/or activity atarget RNA is reduced. The expression, level and/or activity of apolypeptide encoded by the target RNA might also be reduced by a DsiRNAof the instant invention.

In the treatment of a disease or disorder, the DsiRNA can be broughtinto contact with the cells or tissue exhibiting or associated with adisease or disorder. For example, DsiRNA substantially identical to allor part of a target RNA sequence, may be brought into contact with orintroduced into a diseased, disease-associated or infected cell, eitherin vivo or in vitro. Similarly, DsiRNA substantially identical to all orpart of a target RNA sequence may administered directly to a subjecthaving or at risk of developing a disease or disorder.

Therapeutic use of the DsiRNA agents of the instant invention caninvolve use of formulations of DsiRNA agents comprising multipledifferent DsiRNA agent sequences. For example, two or more, three ormore, four or more, five or more, etc. of the presently described agentscan be combined to produce a formulation that, e.g., targets multipledifferent regions of one or more target RNA(s). A DsiRNA agent of theinstant invention may also be constructed such that either strand of theDsiRNA agent independently targets two or more regions of a target RNA.Use of multifunctional DsiRNA molecules that target more then one regionof a target nucleic acid molecule is expected to provide potentinhibition of RNA levels and expression. For example, a singlemultifunctional DsiRNA construct of the invention can target bothconserved and variable regions of a target nucleic acid molecule,thereby allowing down regulation or inhibition of, e.g., differentstrain variants of a virus, or splice variants encoded by a singletarget gene.

A DsiRNA agent of the invention can be conjugated (e.g., at its 5′ or 3′terminus of its sense or antisense strand) or unconjugated to anothermoiety (e.g. a non-nucleic acid moiety such as a peptide), an organiccompound (e.g., a dye, cholesterol, or the like). Modifying DsiRNAagents in this way may improve cellular uptake or enhance cellulartargeting activities of the resulting DsiRNA agent derivative ascompared to the corresponding unconjugated DsiRNA agent, are useful fortracing the DsiRNA agent derivative in the cell, or improve thestability of the DsiRNA agent derivative compared to the correspondingunconjugated DsiRNA agent.

RNAi In Vitro Assay to Assess DsiRNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system canoptionally be used to evaluate DsiRNA constructs. For example, such anassay comprises a system described by Tuschl et al., 1999, Genes andDevelopment, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33,adapted for use with DsiRNA agents directed against target RNA. ADrosophilaextract derived from syncytial blastoderm is used toreconstitute RNAi activity in vitro. Target RNA is generated via invitro transcription from an appropriate plasmid using T7 RNA polymeraseor via chemical synthesis. Sense and antisense DsiRNA strands (forexample 20 uM each) are annealed by incubation in buffer (such as 100 mMpotassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysisbuffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4,2 mM magnesium acetate). Annealing can be monitored by gelelectrophoresis on an agarose gel in TBE buffer and stained withethidium bromide. The Drosophilalysate is prepared using zero totwo-hour-old embryos from Oregon R flies collected on yeasted molassesagar that are dechorionated and lysed. The lysate is centrifuged and thesupernatant isolated. The assay comprises a reaction mixture containing50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10%[vol/vol] lysis buffer containing DsiRNA (10 nM final concentration).The reaction mixture also contains 10 mM creatine phosphate, 10 ug/mlcreatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP,5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. Thefinal concentration of potassium acetate is adjusted to 100 mM. Thereactions are pre-assembled on ice and preincubated at 25° C. for 10minutes before adding RNA, then incubated at 25° C. for an additional 60minutes. Reactions are quenched with 4 volumes of 1.25xPassive LysisBuffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis orother methods known in the art and are compared to control reactions inwhich DsiRNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-32P] CTP, passed over aG50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-32P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by PHOSPHOR IMAGER®(autoradiography) quantitation of bands representing intact control RNAor RNA from control reactions without DsiRNA and the cleavage productsgenerated by the assay.

Methods of Introducing Nucleic Acids, Vectors, and Host Cells

DsiRNA agents of the invention may be directly introduced into a cell(i.e., intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing a cell or organism in a solutioncontaining the nucleic acid. Vascular or extravascular circulation, theblood or lymph system, and the cerebrospinal fluid are sites where thenucleic acid may be introduced.

The DsiRNA agents of the invention can be introduced using nucleic aciddelivery methods known in art including injection of a solutioncontaining the nucleic acid, bombardment by particles covered by thenucleic acid, soaking the cell or organism in a solution of the nucleicacid, or electroporation of cell membranes in the presence of thenucleic acid. Other methods known in the art for introducing nucleicacids to cells may be used, such as lipid-mediated carrier transport,chemical-mediated transport, and cationic liposome transfection such ascalcium phosphate, and the like. The nucleic acid may be introducedalong with other components that perform one or more of the followingactivities: enhance nucleic acid uptake by the cell or other-wiseincrease inhibition of the target RNA.

A cell having a target RNA may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target RNA sequence and the dose of DsiRNAagent material delivered, this process may provide partial or completeloss of function for the target RNA. A reduction or loss of RNA levelsor expression (either RNA expression or encoded polypeptide expression)in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of targetedcells is exemplary. Inhibition of target RNA levels or expression refersto the absence (or observable decrease) in the level of RNA orRNA-encoded protein. Specificity refers to the ability to inhibit thetarget RNA without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS). Inhibition of target RNA sequence(s) by the DsiRNAagents of the invention also can be measured based upon the effect ofadministration of such DsiRNA agents upon measurable phenotypes such astumor size for cancer treatment, viral load/titer for viral infectiousdiseases, etc. either in vivo or in vitro. For viral infectiousdiseases, reductions in viral load or titer can include reductions of,e.g., 50%, 60%, 70%, 80%, 90%, 95% or 99% or more, and are oftenmeasured in logarithmic terms, e.g., 10-fold, 100-fold, 1000-fold,10⁵-fold, 10⁶-fold, 10⁷-fold reduction in viral load or titer can beachieved via administration of the DsiRNA agents of the invention tocells, a tissue, or a subject.

For RNA-mediated inhibition in a cell line or whole organism, expressiona reporter or drug resistance gene whose protein product is easilyassayed can be measured. Such reporter genes include acetohydroxyacidsynthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ),beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), greenfluorescent protein (GFP), horseradish peroxidase (HRP), luciferase(Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivativesthereof. Multiple selectable markers are available that conferresistance to ampicillin, bleomycin, chloramphenicol, gentarnycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, and tetracyclin. Depending on the assay, quantitation of theamount of gene expression allows one to determine a degree of inhibitionwhich is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to acell not treated according to the present invention.

Lower doses of injected material and longer times after administrationof RNA silencing agent may result in inhibition in a smaller fraction ofcells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targetedcells). Quantitation of gene expression in a cell may show similaramounts of inhibition at the level of accumulation of target RNA ortranslation of target protein. As an example, the efficiency ofinhibition may be determined by assessing the amount of gene product inthe cell; RNA may be detected with a hybridization probe having anucleotide sequence outside the region used for the inhibitory DsiRNA,or translated polypeptide may be detected with an antibody raisedagainst the polypeptide sequence of that region.

The DsiRNA agent may be introduced in an amount which allows delivery ofat least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500or 1000 copies per cell) of material may yield more effectiveinhibition; lower doses may also be useful for specific applications.

RNA Interference Based Therapy

As is known, RNAi methods are applicable to a wide variety of genes in awide variety of organisms and the disclosed compositions and methods canbe utilized in each of these contexts. Examples of genes which can betargeted by the disclosed compositions and methods include endogenousgenes which are genes that are native to the cell or to genes that arenot normally native to the cell. Without limitation, these genes includeoncogenes, cytokine genes, idiotype (Id) protein genes, prion genes,genes that expresses molecules that induce angiogenesis, genes foradhesion molecules, cell surface receptors, proteins involved inmetastasis, proteases, apoptosis genes, cell cycle control genes, genesthat express EGF and the EGF receptor, multi-drug resistance genes, suchas the MDR1 gene.

More specifically, a target mRNA of the invention can specify the aminoacid sequence of a cellular protein (e.g., a nuclear, cytoplasmic,transmembrane, or membrane-associated protein). In another embodiment,the target mRNA of the invention can specify the amino acid sequence ofan extracellular protein (e.g., an extracellular matrix protein orsecreted protein). As used herein, the phrase “specifies the amino acidsequence” of a protein means that the mRNA sequence is translated intothe amino acid sequence according to the rules of the genetic code. Thefollowing classes of proteins are listed for illustrative purposes:developmental proteins (e.g., adhesion molecules, cyclin kinaseinhibitors, Wnt family members, Pax family members, Winged helix familymembers, Hox family members, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2,BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS,FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN,NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressorproteins (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF I, NF2, RB I, TP53,and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturasesand hydroxylases, ADP-glucose pyrophorylases, ATPases, alcoholdehydrogenases, amylases, amyloglucosidases, catalases, cellulases,chalcone synthases, chitinases, cyclooxygenases, decarboxylases,dextriinases, DNA and RNA polymerases, galactosidases, glucanases,glucose oxidases, granule-bound starch synthases, GTPases, helicases,hernicellulases, integrases, inulinases, invertases, isomerases,kinases, lactases, lipases, lipoxygenases, lysozymes, nopalinesynthases, octopine synthases, pectinesterases, peroxidases,phosphatases, phospholipases, phosphorylases, phytases, plant growthregulator synthases, polygalacturonases, proteinases and peptidases,pullanases, recombinases, reverse transcriptases, RUBISCOs,topoisomerases, and xylanases).

In one aspect, the target mRNA molecule of the invention specifies theamino acid sequence of a protein associated with a pathologicalcondition. For example, the protein may be a pathogen-associated protein(e.g., a viral protein involved in immunosuppression of the host,replication of the pathogen, transmission of the pathogen, ormaintenance of the infection), or a host protein which facilitates entryof the pathogen into the host, drug metabolism by the pathogen or host,replication or integration of the pathogen's genome, establishment orspread of infection in the host, or assembly of the next generation ofpathogen. Pathogens include RNA viruses such as flaviviruses,picornaviruses, rhabdoviruses, filoviruses, retroviruses, includinglentiviruses, or DNA viruses such as adenoviruses, poxviruses, herpesviruses, cytomegaloviruses, hepadnaviruses or others. Additionalpathogens include bacteria, fungi, helminths, schistosomes andtrypanosomes. Other kinds of pathogens can include mammaliantransposable elements. Alternatively, the protein may be atumor-associated protein or an autoimmune disease-associated protein.

The target gene may be derived from or contained in any organism. Theorganism may be a plant, animal, protozoa, bacterium, virus or fungus.See e.g., U.S. Pat. No. 6,506,559, incorporated herein by reference.

Pharmaceutical Compositions

In certain embodiments, the present invention provides for apharmaceutical composition comprising the DsiRNA agent of the presentinvention. The DsiRNA agent sample can be suitably formulated andintroduced into the environment of the cell by any means that allows fora sufficient portion of the sample to enter the cell to induce genesilencing, if it is to occur. Many formulations for dsNA are known inthe art and can be used so long as the dsNA gains entry to the targetcells so that it can act. See, e.g., U.S. published patent applicationNos. 2004/0203145 A1 and 2005/0054598 A1. For example, the DsiRNA agentof the instant invention can be formulated in buffer solutions such asphosphate buffered saline solutions, liposomes, micellar structures, andcapsids. Formulations of DsiRNA agent with cationic lipids can be usedto facilitate transfection of the DsiRNA agent into cells. For example,cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationicglycerol derivatives, and polycationic molecules, such as polylysine(published PCT International Application WO 97/30731), can be used.Suitable lipids include Oligofectamine, Lipofectamine (LifeTechnologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.),or FuGene 6 (Roche) all of which can be used according to themanufacturer's instructions.

Such compositions typically include the nucleic acid molecule and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” includes saline, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

The compounds can also be administered by transfection or infectionusing methods known in the art, including but not limited to the methodsdescribed in McCaffrey et al. (2002), Nature, 418(6893), 38-9(hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol.,20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J.Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst.Pharm. 53(3), 325 (1996).

The compounds can also be administered by any method suitable foradministration of nucleic acid agents, such as a DNA vaccine. Thesemethods include gene guns, bio injectors, and skin patches as well asneedle-free methods such as the micro-particle DNA vaccine technologydisclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermalneedle-free vaccination with powder-form vaccine as disclosed in U.S.Pat. No. 6,168,587. Additionally, intranasal delivery is possible, asdescribed in, inter alia, Hamajima et al. (1998), Clin. Immunol.Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Pat.No. 6,472,375) and microencapsulation can also be used. Biodegradabletargetable microparticle delivery systems can also be used (e.g., asdescribed in U.S. Pat. No. 6,471,996).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Suchformulations can be prepared using standard techniques. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects istherapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of a nucleic acidmolecule (i.e., an effective dosage) depends on the nucleic acidselected. For instance, if a plasmid encoding a DsiRNA agent isselected, single dose amounts in the range of approximately 1 pg to 1000mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10, 30, 100, or1000 mg may be administered. In some embodiments, 1-5 g of thecompositions can be administered. The compositions can be administeredfrom one or more times per day to one or more times per week; includingonce every other day. The skilled artisan will appreciate that certainfactors may influence the dosage and timing required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present. Moreover, treatment of asubject with a therapeutically effective amount of a protein,polypeptide, or antibody can include a single treatment or, preferably,can include a series of treatments.

It can be appreciated that the method of introducing DsiRNA agents intothe environment of the cell will depend on the type of cell and the makeup of its environment. For example, when the cells are found within aliquid, one preferable formulation is with a lipid formulation such asin lipofectamine and the DsiRNA agents can be added directly to theliquid environment of the cells. Lipid formulations can also beadministered to animals such as by intravenous, intramuscular, orintraperitoneal injection, or orally or by inhalation or other methodsas are known in the art. When the formulation is suitable foradministration into animals such as mammals and more specificallyhumans, the formulation is also pharmaceutically acceptable.Pharmaceutically acceptable formulations for administeringoligonucleotides are known and can be used. In some instances, it may bepreferable to formulate DsiRNA agents in a buffer or saline solution anddirectly inject the formulated DsiRNA agents into cells, as in studieswith oocytes. The direct injection of DsiRNA agents duplexes may also bedone. For suitable methods of introducing dsNA (e.g., DsiRNA agents),see U.S. published patent application No. 2004/0203145 A1.

Suitable amounts of a DsiRNA agent must be introduced and these amountscan be empirically determined using standard methods. Typically,effective concentrations of individual DsiRNA agent species in theenvironment of a cell will be about 50 nanomolar or less, 10 nanomolaror less, or compositions in which concentrations of about 1 nanomolar orless can be used. In another embodiment, methods utilizing aconcentration of about 200 picomolar or less, and even a concentrationof about 50 picomolar or less, about 20 picomolar or less, about 10picomolar or less, or about 5 picomolar or less can be used in manycircumstances.

The method can be carried out by addition of the DsiRNA agentcompositions to any extracellular matrix in which cells can liveprovided that the DsiRNA agent composition is formulated so that asufficient amount of the DsiRNA agent can enter the cell to exert itseffect. For example, the method is amenable for use with cells presentin a liquid such as a liquid culture or cell growth media, in tissueexplants, or in whole organisms, including animals, such as mammals andespecially humans.

The level or activity of a target RNA can be determined by any suitablemethod now known in the art or that is later developed. It can beappreciated that the method used to measure a target RNA and/or theexpression of a target RNA can depend upon the nature of the target RNA.For example, if the target RNA encodes a protein, the term “expression”can refer to a protein or the RNA/transcript derived from the targetRNA. In such instances, the expression of a target RNA can be determinedby measuring the amount of RNA corresponding to the target RNA or bymeasuring the amount of that protein. Protein can be measured in proteinassays such as by staining or immunoblotting or, if the proteincatalyzes a reaction that can be measured, by measuring reaction rates.All such methods are known in the art and can be used. Where target RNAlevels are to be measured, any art-recognized methods for detecting RNAlevels can be used (e.g., RT-PCR, Northern Blotting, etc.). In targetingviral RNAs with the DsiRNA agents of the instant invention, it is alsoanticipated that measurement of the efficacy of a DsiRNA agent inreducing levels of a target virus in a subject, tissue, in cells, eitherin vitro or in vivo, or in cell extracts can also be used to determinethe extent of reduction of target viral RNA level(s). Any of the abovemeasurements can be made on cells, cell extracts, tissues, tissueextracts or any other suitable source material.

The determination of whether the expression of a target RNA has beenreduced can be by any suitable method that can reliably detect changesin RNA levels. Typically, the determination is made by introducing intothe environment of a cell undigested DsiRNA such that at least a portionof that DsiRNA agent enters the cytoplasm, and then measuring the levelof the target RNA. The same measurement is made on identical untreatedcells and the results obtained from each measurement are compared.

The DsiRNA agent can be formulated as a pharmaceutical composition whichcomprises a pharmacologically effective amount of a DsiRNA agent andpharmaceutically acceptable carrier. A pharmacologically ortherapeutically effective amount refers to that amount of a DsiRNA agenteffective to produce the intended pharmacological, therapeutic orpreventive result. The phrases “pharmacologically effective amount” and“therapeutically effective amount” or simply “effective amount” refer tothat amount of an RNA effective to produce the intended pharmacological,therapeutic or preventive result. For example, if a given clinicaltreatment is considered effective when there is at least a 20% reductionin a measurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 20%reduction in that parameter.

Suitably formulated pharmaceutical compositions of this invention can beadministered by any means known in the art such as by parenteral routes,including intravenous, intramuscular, intraperitoneal, subcutaneous,transdermal, airway (aerosol), rectal, vaginal and topical (includingbuccal and sublingual) administration. In some embodiments, thepharmaceutical compositions are administered by intravenous orintraparenteral infusion or injection.

In general, a suitable dosage unit of dsNA will be in the range of 0.001to 0.25 milligrams per kilogram body weight of the recipient per day, orin the range of 0.01 to 20 micrograms per kilogram body weight per day,or in the range of 0.01 to 10 micrograms per kilogram body weight perday, or in the range of 0.10 to 5 micrograms per kilogram body weightper day, or in the range of 0.1 to 2.5 micrograms per kilogram bodyweight per day. Pharmaceutical composition comprising the dsNA can beadministered once daily. However, therapeutic agent may also be dosed indosage units containing two, three, four, five, six or more sub-dosesadministered at appropriate intervals throughout the day. In that case,the dsNA contained in each sub-dose must be correspondingly smaller inorder to achieve the total daily dosage unit. The dosage unit can alsobe compounded for a single dose over several days, e.g., using aconventional sustained release formulation which provides sustained andconsistent release of the dsNA over a several day period. Sustainedrelease formulations are well known in the art. In this embodiment, thedosage unit contains a corresponding multiple of the daily dose.Regardless of the formulation, the pharmaceutical composition mustcontain dsNA in a quantity sufficient to inhibit expression of thetarget gene in the animal or human being treated. The composition can becompounded in such a way that the sum of the multiple units of dsNAtogether contain a sufficient dose.

Data can be obtained from cell culture assays and animal studies toformulate a suitable dosage range for humans. The dosage of compositionsof the invention lies within a range of circulating concentrations thatinclude the ED₅₀ (as determined by known methods) with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range of the compound that includes the IC₅₀ (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsof dsNA in plasma may be measured by standard methods, for example, byhigh performance liquid chromatography.

The pharmaceutical compositions can be included in a kit, container,pack, or dispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a diseaseor disorder caused, in whole or in part, by the expression of a targetRNA and/or the presence of such target RNA (e.g., in the context of aviral infection, the presence of a target RNA of the viral genome,capsid, host cell component, etc.).

“Treatment”, or “treating” as used herein, is defined as the applicationor administration of a therapeutic agent (e.g., a DsiRNA agent or vectoror transgene encoding same) to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has the disease or disorder, a symptom of disease ordisorder or a predisposition toward a disease or disorder, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, the symptoms of the diseaseor disorder, or the predisposition toward disease.

In one aspect, the invention provides a method for preventing in asubject, a disease or disorder as described above, by administering tothe subject a therapeutic agent (e.g., a DsiRNA agent or vector ortransgene encoding same). Subjects at risk for the disease can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the detection of, e.g., viral particles in asubject, or the manifestation of symptoms characteristic of the diseaseor disorder, such that the disease or disorder is prevented or,alternatively, delayed in its progression.

Another aspect of the invention pertains to methods of treating subjectstherapeutically, i.e., alter onset of symptoms of the disease ordisorder. These methods can be performed in vitro (e.g., by culturingthe cell with the DsiRNA agent) or, alternatively, in vivo (e.g., byadministering the DsiRNA agent to a subject).

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target RNAmolecules of the present invention or target RNA modulators according tothat individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

Therapeutic agents can be tested in an appropriate animal model. Forexample, a DsiRNA agent (or expression vector or transgene encodingsame) as described herein can be used in an animal model to determinethe efficacy, toxicity, or side effects of treatment with said agent.Alternatively, a therapeutic agent can be used in an animal model todetermine the mechanism of action of such an agent. For example, anagent can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent can be used in an animal model to determine themechanism of action of such an agent.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow andLane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6thEdition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. Aguide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ.of Oregon Press, Eugene, 2000).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1 Methods Oligonucleotide Synthesis, In Vitro Use

Individual RNA strands were synthesized and HPLC purified according tostandard methods (Integrated DNA Technologies, Coralville, Iowa). Alloligonucleotides were quality control released on the basis of chemicalpurity by HPLC analysis and full length strand purity by massspectrometry analysis. Duplex RNA DsiRNAs were prepared before use bymixing equal quantities of each strand, briefly heating to 100° C. inRNA buffer (IDT) and then allowing the mixtures to cool to roomtemperature.

Oligonucleotide Synthesis, In Vivo Use

Individual RNA strands were synthesized and HPLC purified according tostandard methods (OligoFactory, Holliston, Mass.). All oligonucleotideswere quality control released on the basis of chemical purity by HPLCanalysis and full length strand purity by mass spectrometry analysis.Duplex RNA DsiRNAs were prepared before use by mixing equimolarquantities of each strand, briefly heating to 100° C. in RNA buffer(IDT) and then allowing the mixtures to cool to room temperature.

Cell culture and RNA transfection

HeLa cells were obtained from ATCC and maintained in Dulbecco's modifiedEagle medium (HyClone) supplemented with 10% fetal bovine serum(HyClone) at 37° C. under 5% CO₂. For RNA transfections of FIG. 2, HeLacells were seeded overnight in 6-well plates at a density of 4×10⁵cells/well in a final volume of 2 mL. 24 hours later, cells weretransfected with the DsiRNA duplexes as specified at a finalconcentration of 20 nM using Oligofectamine™ (Invitrogen) and followingthe manufacturer's instructions. Briefly, 84, of a 504 stock solution ofeach DsiRNA was mixed with 2004, of Opti-MEM® I (Invitrogen). In aseparate tube, 124, of Oligofectamine™ was mixed with 484, of Opti-MEM®I. After a 5 minute incubation at room temperature (RT) the DsiRNA andOligofectamine™ aliquots were combined, gently vortexed, and furtherincubated for 20 minutes at RT to allow Dsi_RNA:Oligofectamine™complexes (transfection mixes) to form. Finally, culture medium wasadded to bring each transfection mix to a final volume of 2 mL. After a6 hour incubation, the transfection/culture medium in each well wasreplaced with fresh culture medium and cells were incubated for anadditional 18 hours.

For RNA transfections of FIGS. 3-5, HeLa cells were transfected withDsiRNAs as indicated at a final concentration of 0.1 nM usingLipofectamine™ RNAiMAX (Invitrogen) and following manufacturer'sinstructions. Briefly, 2.54, of a 0.0204 stock solution of each DsiRNAwere mix with 46.5 μL of Opti-MEM I (Invitrogen) and 1 μL ofLipofectamine™ RNAiMAX. The resulting 50 μL mix was added intoindividual wells of 12 well plates and incubated for 20 min at RT toallow Dsi_RNA:Lipofectamine™ RNAiMAX complexes to form. Meanwhile, HeLacells were trypsinized and resuspended in medium at a finalconcentration of 367 cells/μL. Finally, 450 μL, of the cell suspensionwere added to each well (final volume 500 μL) and plates were placedinto the incubator for 24 hours.

RNA Isolation and Analysis, in Vitro Examples

Cells were washed once with 2 mL of PBS, and total RNA was extractedusing RNeasy Mini Kit™ (Qiagen) and eluted in a final volume of 30 μL. 1μg of total RNA was reverse-transcribed using Transcriptor 1^(st) StrandcDNA Kit™ (Roche) and random hexamers following manufacturer'sinstructions. One-thirtieth (0.66 μL) of the resulting cDNA was mixedwith 5 μL, of iQ™ Multiplex Powermix (Bio-Rad) together with 3.33 μL ofH₂O and 1 μL of a 30 μM mix containing 2 sets of primers and probesspecific for human genes HPRT-1 (accession number NM_(—)000194) andSFRS9 (accession number NM_(—)003769) genes:

Hu HPRT forward primer F517 GACTTTGCTTTCCTTGGTCAGHu HPRT reverse primer R591 GGCTTATATCCAACACTTCGTGGG Hu HPRT probe P554Cy5-ATGGTCAAGGTCGCAAGCTTGCTGGT-IBFQ Hu SFRS9 forward primer F569TGTGCAGAAGGATGGAGT Hu SFRS9 reverse primer R712 CTGGTGCTTCTCTCAGGATAHu SFRS9 probe P644 HEX-TGGAATATGCCCTGCGTAAACTGGA-IBFQ

In Vivo Sample Preparation and Injection

DsiRNA was formulated in Invivofectamine™ according to manufacturer'sprotocol (Invitrogen, Carlsbad, Calif.). Briefly, the N/group of miceand body weight of the mice used were determined, then amount of DsiRNAneeded for each group of mice treated was calculated. One ml IVF-oligowas enough for 4 mice of 25 g/mouse at 10 mg/kg dosage. One mg DsiRNAwas added to one ml Invivofectamine™, and mixed at RT for 30 min on arotator. 14 ml of 5% glucose was used to dilute formulated IVF-DsiRNAand applied to 50 kDa molecular weight cutoff spin concentrators(Amicon). The spin concentrators were spun at 4000 rpm for ˜2 hours at 4C until the volume of IVF-DsiRNA was brought down to less than 1 ml.Recovered IVF-DsiRNA was diluted to one ml with 5% glucose and readiedfor animal injection.

Animal Injection and Tissue Harvesting

Animals were subjected to surgical anesthesia by i.p. injection withKetamine/Xylazine. Each mouse was weighed before injection. FormulatedIVF-DsiRNA was injected i.v. at 100 ul/10 g of body weight. After 24hours, mice were sacrificed by CO₂ inhalation. Tissues for analysis werecollected and placed in tubes containing 2 ml RNAlater™ (Qiagen) androtated at RT for 30 min before incubation at 4° C. overnight. Thetissues were stored subsequently at −80° C. until use.

Tissue RNA Preparation and Quantitation

About 50-100 mg of tissue pieces were homogenized in 1 ml QIAzol™(Qiagen) on Tissue Lyser™ (Qiagen). Then total RNA were isolatedaccording to the manufacturer's protocol. Briefly, 0.2 ml Chloroform(Sigma-Aldrich) was added to the QIAzol™ lysates and mixed vigorously byvortexing. After spinning at 14,000 rpm for 15 min at 4° C., aqueousphase was collected and mixed with 0.5 ml of isopropanol. After anothercentrifugation at 14,000 rpm for 10 min, the RNA pellet was washed oncewith 75% ethanol and briefly dried. The isolated RNA was resuspended in100 ul RNase-Free water, and subjected to clean up with RNeasy™ totalRNA preparation kit (Qiagen) or SV 96 total RNA Isolation System(Promega) according to manufacturer's protocol.

First Strand cDNA Synthesis, In Vivo Examples

1 μg of total RNA was reverse-transcribed using Transcriptor 1^(st)Strand cDNA Kit™ (Roche) and oligo-dT following manufacturer'sinstructions. One-fortieth (0.66 μL) of the resulting cDNA was mixedwith 5 μL of IQ Multiplex Powermix (Bio-Rad) together with 3.33 μL ofH₂O and 1 μL, of a 3 μL mix containing 2 sets of primers and probesspecific for mouse genes HPRT-1 (accession number NM_(—)013556) and KRAS(accession number NM_(—)021284) genes:

Mm HPRT forward primer F576 CAAACTTTGCTTTCCCTGGTMm HPRT reverse primer R664 CAACAAAGTCTGGCCTGTATC Mm HPRT probe P616Cy5-TGGTTAAGGTTGCAAGCTTGCTGGTG-IBFQ Mm KRAS forward primer F275CTTTGTGGATGAGTACGACC Mm KRAS reverse primer R390 CACTGTACTCCTCTTGACCTMm KRAS probe P297 FAM-ACGATAGAGGACTCCTACAGGAAACAAGT-IBFQ

Quantitative RT-PCR

A CFX96 Real-time System with a C1000 Thermal cycler (Bio-Rad) was usedfor the amplification reactions. PCR conditions were: 95° C. for 3 min;and then cycling at 95° C., 10 sec; 55° C., 1 min for 40 cycles. Eachsample was tested in triplicate. For HPRT Examples, relative HPRT mRNAlevels were normalized to SFRS9 mRNA levels and compared with mRNAlevels obtained in control samples treated with the transfection reagentplus a control mismatch duplex, or untreated. For KRAS examples,relative KRAS mRNA levels were normalized to HPRT-1 mRNA levels andcompared with mRNA levels obtained in control samples from mice treatedwith 5% glucose. Data were analyzed using Bio-Rad CFX Manager version1.0 (in vitro Examples) or 1.5 (in vivo Example) software.

Example 2 Efficacy of DsiRNA Agents Possessing DNA Duplex Extensions

DsiRNA agents possessing DNA duplex extensions were examined forefficacy of sequence-specific target mRNA inhibition. Specifically,HPRT-targeting DsiRNA duplexes possessing RNA-extended, DNA-extended ormixed DNA- and RNA-extended structures were transfected into HeLa cellsat a fixed concentration of 20 nM and HPRT expression levels weremeasured 24 hours later (FIGS. 2A and 2B). Transfections were performedin duplicate, and each duplicate was assayed in triplicate for HPRTexpression by qPCR. Under these conditions (20 nM duplexes,Oligofectamine transfection), HPRT gene expression was reduced by 30-50%by duplexes 1 through 6. Duplex 6 contained DNA substitutions whichformed a 10 bp (base pair) region starting at the 5′ end of the guide(antisense) strand and gave a final length configuration of 33/35mer.Duplex 7 was identical in length and sequence to duplex 6, but containedonly 4 DNA nucleoside modifications in the positions indicated in FIG.2B. Surprisingly, duplex 7 reduced HPRT expression significantly lessthan duplex 6, suggesting that Dicer recognizes the extended RNA regionbetween the DNA bases and cleaves the duplex into alternate species ofsiRNAs. It was also observed that phosphorothioate modification ofDNA:DNA-extended regions of DsiRNA (duplex 8) was capable of abolishingthe RNA inhibitory activity of DNA-extended DsiRNA agents. It is likelythat activity of the duplex 8 agent can be restored by sufficientsubstitution of PS-DNA moieties with unmodified DNA moieties. Indeed,such “add-back” of unmodified DNA residues to such a duplex underscoresan advantage of the invention—the agents of the invention can be made tocarry more modifications than non-extended agents while still retainingRNA inhibitory activity, which is an important development in view ofthe issues that presence of tandem and/or tightly-spaced modifiedresidues can cause (here, complete abolishment of RNA inhibitoryactivity when tandem, base paired PS-DNAs are present in duplex 8).

Example 3 Dose-Response Comparison of 27/29mer Duplexes

To test the efficacy of a DNA duplex-extended DsiRNA at reducedconcentrations, a modified duplex targeting HPRT was compared to anoptimized 27/29mer duplex in a dose response series of experiments at10.0 nanomolar (nM), 1.0 nanomolar (nM) and 100 picomolar (100 pM or 0.1nM) concentrations for knockdown of HPRT mRNA levels in HeLa cells.Duplex DsiRNA 1 was a derivative of a 25/27mer DsiRNA duplex previouslyreported as active (HPRT-1, Rose et al. NAR 2005, Collingwood et al.2008); however, the present duplex (#1) contained an insertion of twobases in each strand that extended the oligonucleotide duplex to a27/29mer. Duplex 2 was identical in sequence to duplex 1, but the twobase pair insertion and two additional nucleosides of the guide strand(antisense sequence) were synthesized as DNA. Thus, duplex 2 terminatedin 4 DNA by (base pairs) at the 5′ end of the guide strand, in contrastto previously reported two base DNA substitutions at the 3′ end of thepassenger (sense) strand (Rose et al, 2005). Duplex 3 (MM control) wasderived from the optimized HPRT-1 duplex, but synthesized withmismatches in relation to the target RNA sequence. The base compositionand chemical modification of each strand and the base sequences andoverhang or blunt structure at the ends of duplex 3 were held constantrelative to the optimized HPRT-1 duplex in order to control fornon-targeted chemical effects (see FIG. 5). Baseline HPRT expression inuntreated cells was also measured (“C” of FIG. 3A).

Putative Dicer processing products of duplexes 1 and 2 were identical(see FIG. 1A) to one another, and to the Dicer processing products ofduplexes shown in FIG. 2B. At 10 nM and 1 nM transfectionconcentrations, both duplexes 1 and 2 reduced HPRT RNA levels by atleast 95%, suggesting that the addition of the double-stranded DNA atthe end of Duplex 2 did not interfere with Dicer binding and processingof the duplex into a structure competent to load and direct RISCactivity against HPRT mRNA. At 100 pM, duplex 2 reduced HPRT expressionby 90% and appeared more than 2-fold more active than duplex 1, whichreduced HPRT by approximately 75%.

Example 4 Comparison of Duplexes Extended by Two, Six and Eight DNA BasePairs

To investigate the length of double stranded DNA extension that might beintroduced into a DsiRNA agent while still enhancing efficacy and/orduration of effect of such a DNA duplex-extended DsiRNA in comparison toa DsiRNA agent having a corresponding length of extended double strandedRNA, a series of double stranded nucleic acids were generated and testedwith two base pair, six base pair and eight base pair extensions (thenominal length of such extensions also includes penultimate and ultimatedeoxyribonucleotide residues of the 3′ terminus of the sense strand thatbase pair with cognate deoxyribonucleotide residues of the 5′ terminusof the antisense strand, resulting in the “DNA 4 bp” duplex #4, the “DNA8 bp” duplex #5 and the “DNA 10 bp” duplex #6 of FIGS. 4A-4D).Inhibition of gene expression by duplex 3, a 33/35mer comprising anextension comprising a two base pair DNA:RNA double stranded region anda six base pair RNA:RNA double stranded region (see FIGS. 4A-4D) wasreduced relative to duplex 1 (a 27/29mer having a two base pair RNA:RNAdouble stranded extension) and duplex 2 (a 31/33mer having a six basepair RNA:RNA double stranded extension), consistent with previousreports that increasing RNA duplex length lowered RNAi activity.Notably, RNA:RNA-extended duplexes 1, 2 and 3 all showed reducedactivity relative to corresponding duplexes 4, 5, and 6, which possesseddouble stranded DNA:DNA extensions. The greatest difference in activitybetween the RNA insert/DNA series and the DNA series was observed at 100pM, but was still detectable at 10 pM (FIGS. 4B and 4C).

The duplexes compared in FIG. 4 were designed to 1) enhance negativeeffects of promiscuous processing of Dicer-substrate duplexes, if itoccurred, and 2) to eliminate the possibility of RNase H-mediatedcleavage of the HPRT mRNA. Duplexes processed in a way that did notyield a canonical 19-23 base long RNA strand, beginning with the 3′-endof the guide (antisense) strands shown in FIG. 4B, would be less likelyto direct RISC-mediated reduction in HPRT target mRNA levels.Promiscuous processing of long RNA duplexes (e.g., duplex 3 of FIG. 4D)would yield guide strands that contained mismatches in the seed region,thus reducing RISC activity against the target. Promiscuous processingcould also yield RISC loaded with passenger (sense) oligonucleotides.Duplex 3 was significantly less active than shorter RNA species at lowerconcentrations, and was likely processed into less active siRNA species.

If long duplexes containing DNA (e.g., duplex 6 of FIG. 4D) weredifferentially degraded and/or incorrectly processed, single strandedoligonucleotides containing up to ten bases of antisense DNA couldresult. In theory, this DNA portion could activate RNase H to cleave acomplementary target mRNA. The DNA portions of duplexes 4, 5, and 6 didnot match HPRT mRNA, and thus could not be responsible for the observedreduction in HPRT mRNA. Differential degradation of duplexes prior tocellular uptake, processing by Dicer, or before loading into RISC couldalso have caused an observed difference in HPRT reduction. Duplex 3contained an internal substitution of two DNA nucleosides to control forthis effect (bases 9 and 10, counting from the 3′ end of the passengerstrand). If DNA substitutions increased duplex activity by simplystabilizing the duplex against nuclease degradation, duplex 3 shouldhave been more stable than duplexes 1 or 2, and potentially as stable asduplex 4. Instead, duplex 3 reduced HPRT target mRNA levels lesseffectively than duplexes 1, 2, and 4, indicating that the enhancingeffect seen when double stranded DNA:DNA regions were introduced was notsimply attributable to enhanced resistance to nuclease degradation. Bysimilar rationale, if DNA base pairs had caused a significantstabilization of the tested duplexes, then increased DNA base pairlength should have resulted in progressively enhanced activity acrossduplexes 4 through 6. However, such progressively increasing DNA lengthsdid not increase duplex activity.

In view of the above results, it was concluded that DsiRNA agentspossessing double stranded DNA:DNA extended regions of two to ten basepairs (where such extensions were located in the region of the sensestrand that was 3′ of the projected Dicer cleavage site andcorresponding region of the antisense strand that was 5′ of theprojected Dicer cleavage site) constituted effective, and in certaincases, enhanced, RNA inhibitory agents.

Example 5 Enhanced Efficacy of Double Stranded DNA:DNA-Extended Duplexesat Low Concentration

A series of modified duplexes of increasing length was evaluated forreduction of HPRT mRNA expression at a fixed concentration of 100 pM.Duplex 1 of FIGS. 5A and 5B was an optimized 25/27mer Dicer-substratecontaining chemical modifications, a two-base overhang at the 3′-end ofthe guide (antisense) strand and two DNA substitutions and a blunt endat the 3′-end of the passenger (sense) strand (Collingwood et al. 2008).Bases non-complementary to HPRT mRNA were inserted two bases at a timeas either RNA (duplexes 2 through 5) or DNA (duplexes 6 through 9; seeFIGS. 5A and 5B), increasing total duplex configurations from 27/29mersto 33/35mers.

Duplex 1 was more effective at reducing HPRT mRNA levels than any other“optimized” duplex extended by the addition of RNA base pairs (duplexes2 through 5; FIG. 5A), supporting the concept that longer duplexes werelikely processed into one or more less active guide species. Allduplexes extended by the addition of DNA base pairs (duplexes 6 though9; FIG. 5A) were significantly more active than duplexes 2 though 5, andapproximately equal in activity to duplex 1.

Duplexes 6 through 9 were indistinguishable in their degree of HPRT mRNAreduction. Thus, the DNA base pair regions were not exerting anuclease-resistance property that increased RNAi activity (compareduplexes 2 through 5 to each other and to duplex 1). Surprisingly,increasing the length of the DNA portion also did not negatively impactHPRT reduction. All DNA duplexes had equivalent activity and had greateractivity than comparable RNA-based duplexes. These results indicatedthat the DNA insertions of duplexes 2 through 5 limited Dicer activityto production of the canonical guide strand processed out of optimizedduplex 1.

Example 6 Efficacy of Left-Extended DsiRNA Agents, Including DsiRNAAgents Harboring Mismatches (“DsiRNAmms”)

To examine whether effective DsiRNA agents can also possess DNA:DNAextensions in the reverse (“flipped”) orientation as the above-described“right extended” DsiRNA agents, “left extended” DsiRNA agents weresynthesized and tested for inhibitory efficacy via methods as describedabove. Such “left extended” DsiRNA agents (in the instant case, 30/28meragents possessing a 5 base pair DNA:DNA extension formed between the 5′terminal region of the sense strand and 3′ terminal region of theantisense strand, as shown in FIG. 7) were tested for target RNAinhibitory efficacy in direct comparison with corresponding 28/30mer“right extended” DsiRNA agents. Surprisingly, “left extended” agentswere observed to be more potent than “right extended” agents in theirinhibition of KRAS target mRNA levels. Also surprising was the fact thatmismatch residues could be introduced into both “left extended” and“right extended” DNA:DNA extended DsiRNA agents at certain positions butnot others, while retaining inhibition efficacy of such agents.Specifically, DNA:DNA extended “DsiRNAmm” agents (as used herein, theterm “DsiRNAmm agent” indicates a DsiRNA agent comprising one or moremismatched base pairs that are positioned at a location other than thetwo terminal nucleotide residues of either end of either strand) weresynthesized to possess mismatched base pairs at the following positions:12 alone, 14 alone, 16 alone, 14 and 18 together and 12 and 16 together(starting from position 1 as the 5′ terminal antisense residue of theprojected post-Dicer cleaved DsiRNAmm agent). As shown in FIGS. 8 and 9,DsiRNAmm agents possessing mismatched residues at position 14 alone,position 16 alone and at both positions 14 and 18, were all effectiveinhibitory agents. Surprisingly, left-extended forms of both “parent”DsiRNAs and DsiRNAmms were initially identified to possess greaterinhibition efficacy (at 100 pM transfection levels in HeLa cells) thanright extended forms for parent DsiRNA agents and for DsiRNAmm agentshaving mismatched residues at position 14 alone, position 16 alone andat both positions 14 and 18 of the antisense strand (when positions arenumbered in the 3′ direction (meaning from 5′ to 3′) starting fromposition 1 at the 5′ terminal antisense residue of the predictedpost-Dicer cleaved DsiRNA or DsiRNAmm agent; see FIG. 8). With theexception of the DsiRNAmm agent possessing mismatched residues at bothpositions 14 and 18 of the antisense strand, the greater efficacy at 100pM which was initially observed for left-extended as compared toright-extended DsiRNA or DsiRNAmm agents was reproducible (FIG. 9).

Example 7 Location of Phosphorothioate Modifications and DNA ResiduesWithin Effective DsiRNA Agents

To test whether DNA:DNA extended sequences of the invention provideextra residues within a DsiRNA agent upon which advantageousmodifications might be placed while retaining inhibitory efficacy of theDsiRNA agent, the robustness of DsiRNA agents harboring multiplephosphorothioate-modified bases was examined. Prior studies ofphosphorothioate modified siRNA agents have revealed that such agentscan be cytotoxic to cells when multiple phosphorothioates are present(Amarzguioui et al. Nucleic Acids Research, 31: 589-595), and some siRNAagents possessing phosphorothioate modifications at or near the 5′ endof the antisense strand have been observed to have reduced inhibitoryactivity. As shown in FIG. 10, the DsiRNA agents “DNA 6 bp(2PS)” and“DNA 6 bp(4PS)” exhibited similar target mRNA (HPRT) inhibitoryefficacies, demonstrating that the DNA-extended duplex regions of thesemolecules can be extensively modified without detrimental impact uponthese molecules' target RNA inhibitory efficacy.

The patterning of deoxyribonucleotides at or near the projected 5′ Dicercleavage site of the antisense strand within “right-extended” DsiRNAagents of the invention was also examined. As shown in FIG. 10, DsiRNAagents possessing antisense strand deoxyribonucleotides extending fromthe 5′ terminus of the antisense strand all the way to the locationadjacent to the 5′ terminal nucleotide of the post-Dicer cleavedantisense strand (see agents DP1055P/DP1057G and DP1058P/DP1060G) wereeffective RNA interference agents. Results for the DP1055P/DP1057G andDP1058P/DP1060G DsiRNA agents were unexpected, as it was previouslythought that termination of deoxyribonucleotide inclusion within the 5′end region of the antisense strand should occur at a location 5′ withinthe antisense strand of the most 3′ Dicer cleavage site within the sensestrand (see agents DP1055P/DP1056G and DP1058P/DP1059G). As also shownin FIG. 10, a left-extended DsiRNA agent was observed to be an effectiveinhibitory agent, while inclusion of a U:G mismatch within the “seed”region of the antisense strand of this DsiRNA agent was observed tocause a modestly diminished level of inhibitory activity.

The effect of strand-weighted patterns of phosphorothioate modificationof “right-extended” DsiRNA agents of the invention was also examined.The phosphorothioate-modified oligonucleotide strands of“right-extended” DsiRNA agents “DNA 6 bp(2PS)” and “DNA 6 bp(4PS)” werereassembled to create the DP1061P/DP1064G and DP1062G/DP1063P DsiRNAagents shown in FIG. 11. Surprisingly, the DP1062G/DP1063P duplex, whichpresents four phosphorothioate modified deoxyribonucleotides on thepassenger strand and only two phosphorothioate modifieddeoxyribonucleotides on corresponding guide strand residues, performedas well as or better than the “DNA 6 bp(2PS)” agent, whereas theDP1061P/DP1064G duplex, which harbors four phosphorothioate modifieddeoxyribonucleotides on the guide strand and only two phosphorothioatemodified deoxyribonucleotides on corresponding passenger strandresidues, was not as effective an inhibitory molecule. Such resultssuggest that for the extended regions of DsiRNAs of the invention,phosphorothioate modification patterns that weight such modifications onthe passenger strand relative to the guide strand might retain greatestefficacy relative to oppositely weighted patterns.

Example 8 Comparison of Duplexes Extended by Five, Ten and Twelve DNA orRNA Base Pairs

To investigate further the impact of structural extensions of DsiRNAs,several series of “right-extended” DsiRNAs targeting the KRAS transcriptwere generated and assessed for target knockdown efficacy in vitro. FIG.12 depicts the structures of a series of “right-extended” DsiRNAstargeting the “KRAS-200” site within the KRAS transcript. The firstduplex of FIG. 12 (“DP1174P/DP1175G” or duplex “01” of correspondingdata FIG. 13) is a 25/27mer DsiRNA possessing deoxyribonucleotides atonly the ultimate and penultimate 3′-terminal residues of the passengerstrand. The second duplex of FIG. 12 (“DP1200P/DP1201G” or duplex “02”of corresponding data FIG. 13) is a 25/27mer DsiRNA possessingdeoxyribonucleotides at all passenger strand residues located 3′ of thepassenger strand projected Dicer cleavage site and at all guide strandresidues located 5′ of the guide strand projected Dicer cleavage siteshown. The third duplex of FIG. 12 (“DP1202P/DP1203G” or duplex “03” ofcorresponding data FIG. 13) is a 30/32mer DsiRNA possessing a five basepair ribonucleotide sequence insertion relative to the “DP1174P/DP1175G”duplex, as shown (boxed region of the third duplex of FIG. 12). Thefourth duplex of FIG. 12 (“DP1204P/DP1205G” or duplex “04” ofcorresponding data FIG. 13) is a 30/32mer DsiRNA possessing a five basepair deoxyribonucleotide sequence insertion relative to the“DP1200P/DP1201G” duplex, as shown (boxed region of the fourth duplex ofFIG. 12). The fifth duplex of FIG. 12 (“DP1206P/DP1207G” or duplex “05”of corresponding data FIG. 13) is a 35/37mer DsiRNA possessing a tenbase pair ribonucleotide sequence insertion relative to the“DP1174P/DP1175G” duplex, as shown (boxed region of the fifth duplex ofFIG. 12). The sixth duplex of FIG. 12 (“DP1208P/DP1209G” or duplex “06”of corresponding data FIG. 13) is a 35/37mer DsiRNA possessing a tenbase pair deoxyribonucleotide sequence insertion relative to the“DP1200P/DP1201G” duplex, as shown (boxed region of the sixth duplex ofFIG. 12).

FIG. 13 shows KRAS target gene inhibitory efficacy results for theKRAS-200 site targeting DsiRNAs presented in FIG. 12. As shown in FIG.13, DsiRNAs possessing RNA duplex or DNA duplex extensions of five oreven ten base pairs in length retained robust inhibitory efficacy invitro (the experiments of FIG. 13 were performed in HeLa cells andinvolved treatment with 0.1 nM DsiRNA for 24 hours, in duplicate, usingRNAiMAX; “13” and “14” correspond to results obtained using RNAiMAXalone and obtained for untreated cells, respectively; multiplexexperiments were performed to assess both KRAS and HPRT1 levels).

FIG. 14 depicts the structures of a series of “right-extended” DsiRNAstargeting the “KRAS-909” site within the KRAS transcript. The firstduplex of FIG. 14 (“DP1188P/DP1189G”) is a 25/27mer DsiRNA possessingdeoxyribonucleotides at only the ultimate and penultimate 3′-terminalresidues of the passenger strand. The second duplex of FIG. 14(“DP1210P/DP1211G”) is a 25/27mer DsiRNA possessing deoxyribonucleotidesat all passenger strand residues located 3′ of the passenger strandprojected Dicer cleavage site and at all guide strand residues located5′ of the guide strand projected Dicer cleavage site shown. The thirdduplex of FIG. 14 (“DP1212P/DP1213G”) is a 30/32mer DsiRNA possessing afive base pair ribonucleotide sequence insertion relative to the“DP1188P/DP1189G” duplex, as shown (boxed region of the third duplex ofFIG. 14). The fourth duplex of FIG. 14 (“DP1214P/DP1215G”) is a 30/32merDsiRNA possessing a five base pair deoxyribonucleotide sequenceinsertion relative to the “DP1210P/DP1211G” duplex, as shown (boxedregion of the fourth duplex of FIG. 14). The fifth duplex of FIG. 14(“DP1216P/DP1217G”) is a 35/37mer DsiRNA possessing a ten base pairribonucleotide sequence insertion relative to the “DP1188P/DP1189G”duplex, as shown (boxed region of the fifth duplex of FIG. 14). Thesixth duplex of FIG. 14 (“DP1218P/DP1219G”) is a 35/37mer DsiRNApossessing a ten base pair deoxyribonucleotide sequence insertionrelative to the “DP1210P/DP1211G” duplex, as shown (boxed region of thesixth duplex of FIG. 14).

FIG. 15 shows KRAS target gene inhibitory efficacy results for theKRAS-909 site targeting DsiRNAs presented in FIG. 14. As shown in FIG.15, while 25/27mer DsiRNAs “1188P/1189G” and 30/32mer “1212P/1213G”showed slightly greater target RNA inhibitory efficacies than otherDsiRNAs examined, DsiRNAs possessing RNA duplex or DNA duplex extensionsof five or even ten base pairs in length exhibited robust inhibitoryefficacies in vitro. (the experiments of FIG. 15 were performed in HeLacells and involved treatment with 0.1 nM DsiRNA for 24 hours, induplicate, using RNAiMAX; multiplex experiments were performed to assessboth KRAS and HPRT1 levels).

Example 9 Effect of Numerous Phosphorothioate Modifications within“Extended” DsiRNAs

FIG. 10 demonstrates that deoxyribonucleotide extension of DsiRNAmolecules can provide a surface upon which phosphorothioate modificationcan be performed with little, if any, impact upon target transcriptinhibitory efficacy of the phosphorothioate-modified DsiRNA. FIGS. 16-18depict DsiRNA molecules synthesized for purpose of testing whether evenmore extensive levels of phosphorothioate modification can be toleratedwithin extended (here, “right-extended”) DsiRNAs. Specifically, FIG. 16shows a series of “KRAS-249” site-targeting DsiRNAs, wherein:

-   -   the first duplex (“K249M”) is a 25/27mer possessing        deoxyribonucleotides at only the penultimate and ultimate        residues at the 3′-terminus of the passenger strand, has no        phosphorothioate modifications and has a pattern of 2′-O-Methyl        modification as shown (underlined residues indicate 2-O-Methyl        modified residues).    -   the second duplex (“K249D”) is a 31/33mer possessing a total of        eight deoxyribonucleotide base pairs positioned at the        3′-terminus of the passenger strand/5′-terminus of the guide        strand, having no phosphorothioate modifications and having a        pattern of 2′-O-Methyl modification as shown.    -   the third and fourth duplexes (“K249DNA8” and “K249DNA8p”) are        33/35mers possessing exclusively deoxyribonucleotides at all        passenger strand residues positioned 3′ of the projected Dicer        cleavage site shown and at all residues of the guide strand        located 5′ of the projected Dicer cleavage site shown.        2′-O-Methyl modification patterns were the same as used for the        “K249D” DsiRNA described above. The fourth duplex (“K249DNA8p”)        possesses phosphorothioate modifications at all nucleotides of        the eight base pairs comprising the 3′ terminus of the passenger        strand/5′ terminus of the guide strand.    -   the fifth and sixth duplexes (“K249DNA12” and “K249DNA12p”) are        37/39mers possessing exclusively deoxyribonucleotides at all        passenger strand residues positioned 3′ of the projected Dicer        cleavage site shown and at all residues of the guide strand        located 5′ of the projected Dicer cleavage site shown.        2′-O-Methyl modification patterns were the same as used for the        “K249D” DsiRNA described above. The sixth duplex (“K249DNA12p”)        possesses phosphorothioate modifications at all nucleotides of        the twelve base pairs comprising the 3′ terminus of the        passenger strand/5′ terminus of the guide strand.

FIG. 17 shows a series of “KRAS-516” site-targeting DsiRNAs, wherein:

-   -   the first and second duplexes (“K516DNA8” and “K516DNA8p”) are        33/35mers possessing exclusively deoxyribonucleotides at all        passenger strand residues positioned 3′ of the projected Dicer        cleavage site shown and at all residues of the guide strand        located 5′ of the projected Dicer cleavage site shown.        2′-O-Methyl modified nucleotides are shown as underlined        residues. The second duplex (“K516DNA8p”) possesses        phosphorothioate modifications at all nucleotides of the eight        base pairs comprising the 3′ terminus of the passenger strand/5′        terminus of the guide strand. Notably, a four base pair        deoxyribonucleotide sequence of K249 was introduced into these        extended DsiRNAs (see boxed region labeled as “K249”).    -   the third and fourth duplexes (“K516DNA12” and “K516DNA12p”) are        37/39mers possessing exclusively deoxyribonucleotides at all        passenger strand residues positioned 3′ of the projected Dicer        cleavage site shown and at all residues of the guide strand        located 5′ of the projected Dicer cleavage site shown.        2′-O-Methyl modification patterns were the same as used for the        “K516DNA8” and “K516DNA8p” DsiRNAs described above. The fourth        duplex (“K516DNA12p”) possesses phosphorothioate modifications        at all nucleotides of the twelve base pairs comprising the 3′        terminus of the passenger strand/5′ terminus of the guide        strand. As for “K516DNA8” and “K516DNA8p” DsiRNAs, a four base        pair deoxyribonucleotide sequence of K249 was used introduced        into these extended DsiRNAs (see boxed region labeled as        “K249”).

FIG. 18 shows a series of “KRAS-909” site-targeting DsiRNAs, wherein:

-   -   the first and second duplexes (“K909DNA8” and “K909DNA8p”) are        33/35mers possessing exclusively deoxyribonucleotides at all        passenger strand residues positioned 3′ of the projected Dicer        cleavage site shown and at all residues of the guide strand        located 5′ of the projected Dicer cleavage site shown.        2′-O-Methyl modified nucleotides are shown as underlined        residues. The second duplex (“K909DNA8p”) possesses        phosphorothioate modifications at all nucleotides of the eight        base pairs comprising the 3′ terminus of the passenger strand/5′        terminus of the guide strand. A four base pair        deoxyribonucleotide sequence of K249 was also introduced into        these extended DsiRNAs (see boxed region labeled as “K249”).    -   the third and fourth duplexes (“K909DNA12” and “K909DNA12p”) are        37/39mers possessing exclusively deoxyribonucleotides at all        passenger strand residues positioned 3′ of the projected Dicer        cleavage site shown and at all residues of the guide strand        located 5′ of the projected Dicer cleavage site shown.        2′-O-Methyl modification patterns were the same as used for the        “K909DNA8” and “K909DNA8p” DsiRNAs described above. The fourth        duplex (“K909DNA12p”) possesses phosphorothioate modifications        at all nucleotides of the twelve base pairs comprising the 3′        terminus of the passenger strand/5′ terminus of the guide        strand. As for “K909DNA8” and “K909DNA8p” DsiRNAs, a four base        pair deoxyribonucleotide sequence of K249 was used introduced        into these extended DsiRNAs (see boxed region labeled as        “K249”).

The extended DsiRNAs shown in FIGS. 16-18 were tested for KRAS targettranscript inhibitory efficacy in vitro. Data from such experiments isshown in FIG. 19. Surprisingly, both 33/35mer and 37/39mer DNA-extendedDsiRNAs exhibited significant inhibitory efficacies (refer to “DNA8” and“DNA12” results for each of KRAS-249, 516 and 909 target sites in FIG.19); however, extensive phosphorothioate modification of theDNA-extended region of these DsiRNAs—positioned on both strands of theextended region—reduced inhibitory efficacies (see “DNA8p” and “DNA12p”results for each of KRAS-249, 516 and 909 target sites). Thus, eventhough a pattern of two or even four successive DNA base pairs harboringphosphorothioate modifications of both strands was observed to showlittle or no impact upon the efficacy of a DNA-extended DsiRNA (see FIG.10), FIG. 19 shows that insertion of eight or twelve successivephosphorothioate-modified deoxyribonucleotide base pairs(phosphorothioate modified on both strands) within the “extended”DsiRNAs of the invention can reduce the inhibitory efficacy of such“extended” DsiRNAs.

In spite of the above results, it is noted that positioning ofphosphorothioate modifications on only one strand of the double-strandedextended regions of the extended DsiRNAs of the instant invention hasbeen shown to allow for introduction of longer runs of phosphorothioatemodification with no significant loss of efficacy. For example,inclusion of as many as 15 consecutive phosphorothioate modificationsupon only one strand of the extended region of an extended DsiRNA hasbeen shown to be tolerated without significant loss of efficacy of sucha modified extended DsiRNA (data not shown). Thus, even though theresults of FIG. 19 show the impact of including long tracts ofphosphorothioate modification upon both strands of the extended DsiRNAsof the invention, on the whole, the DNA-containing extended region(s) ofthe DsiRNAs of the instant invention have been demonstrated to provide astructure upon which extensive advantageous modifications (e.g.,phosphorothioate, 2′-O-Methyl or other modification(s) capable ofenhancing stability, delivery, efficacy and/or potency of the extendedDsiRNAs of the invention) can be introduced without negativelyimpacting, e.g., the inhibitory efficacy of the extended DsiRNAs of theinvention.

Example 10 Relative Effect of Position and Number of Mismatches WithinNon-Seed Regions of DsiRNAs

In above Example 6, it was demonstrated that introduction of mismatcheswithin the extended DsiRNAs of the invention could create extended“DsiRNAmm” agents that possess inhibitory efficacies similar to those ofDsiRNAs possessing sequences that are perfectly complementary to targetsequence. Notably, it was observed in FIGS. 7-9 that, of the non-seedregion mismatch positions examined, the mismatch position (“position12”) that impacted efficacy the most was also the position located inclosest proximity to the projected Ago2 cleavage site of the targetstrand sequence. Further to these results, the effect of introducingsequence mismatches within 25/27mer DsiRNA nucleotides at non-seedregion positions substantially removed from the projected Ago2 cleavagesite was examined. FIG. 20 shows the structures of a series of 25/27merDsiRNAs that were synthesized to assess the impact of introducing one ormore mismatch residues (noting that for the DsiRNAmm molecules of FIG.20, mismatches were relative to target sequence only, and not withrespect to corresponding DsiRNA passenger strand sequence residues; itis further noted that a “target-mismatched” nucleotide or residue isdefined for purpose of the invention as a guide strand nucleotide thatforms a mismatch relative to target nucleotide sequence, but that is notnecessarily mismatched relative to a corresponding DsiRNA passengerstrand sequence nucleotide), with such mismatches starting from eitherthe 3′ terminus of the guide strand, or starting from the guide strandposition that is complementary to the 5′ terminal residue of thepassenger strand. As shown in FIG. 21, the 3′ terminal region of theguide strand of the tested 25/27mer DsiRNA surprisingly toleratedintroduction of one or more target-mismatched nucleotides. Indeed,introduction of between one and three target-mismatched nucleotidescommencing from the 3′ terminus of the guide strand of the tested DsiRNAelicited no statistically significant impact upon target inhibitionefficacy (see duplexes DP1301P/DP1303G, DP1301P/DP1304G andDP1305P/DP1306G), while introduction of four, five or even sixtarget-mismatched nucleotides commencing from the 3′ terminus of theguide strand of the tested DsiRNA still resulted in a DsiRNA thatretained significant inhibitory activity (see duplexes DP1307P/DP1308G,DP1309P/DP1310G and DP1311P/DP1312G). Introduction of target-mismatchedresidues within the guide strand that commenced from the guide strandposition that is complementary to the 5′ terminal residue of the DsiRNApassenger strand yielded results consistent with those observed forintroduction of target-mismatched nucleotides commencing from the 3′terminus of the guide strand. Specifically, introduction of between oneand four target-mismatched nucleotides commencing from the guide strandposition that is complementary to the 5′ terminal residue of the DsiRNApassenger strand impacted DsiRNA inhibitory efficacy to approximatelythe same extent as observed for introduction of between three and sixtarget-mismatched nucleotides commencing from the 3′ terminus of theguide strand (consistent with the observed tolerance for the ultimateand penultimate nucleotides of the 3′-terminal guide strand sequence—aswell as the guide strand position complementary to the 5′ terminalresidue of the DsiRNA passenger strand—to target-mismatchednucleotides).

Example 11 In Vivo Efficacy of DsiRNA Agents, Single Dose Results

DsiRNA agents possessing DNA duplex extensions were examined for in vivoefficacy of sequence-specific target mRNA inhibition. Specifically,unmodified KRAS-targeting DsiRNA “K249” of FIG. 20 (“DP1301P/DP1302G”duplex), 2′-O-Methyl-modified KRAS-targeting DsiRNA “K249M” (firstduplex of FIGS. 16) and 2′-O-Methyl-modified “right extended”KRAS-targeting DsiRNA “K249D” (second duplex of FIG. 16; denoted as“K249DNA” in FIGS. 22-25) were formulated in Invivofectamine™ andinjected i.v. into CD1 mice at 10 mg/kg. Expression of KRAS in liver,kidney, spleen and lymph node tissues was measured 24 hourspost-injection (FIGS. 22-25, respectively; each bar presents resultsobtained for four mice per treatment group), with real-time PCR (RT-PCR)performed in triplicate to assess KRAS expression. Under theseconditions, “right extended” DsiRNA “K249DNA” exhibited statisticallysignificant levels of KRAS target gene inhibition in all tissuesexamined. Specific KRAS percent inhibition levels observed in such“K249DNA”-treated tissues and p-values associated with theseobservations were: liver (55%-87%, mean 71%, p=0.010), spleen (92%-98%,mean 94%, P<0.001), kidney (19%-53%, mean 35%, P=0.009) and lymph nodes(47%-81%, mean 59%, P=0.001). Thus, the in vivo efficacy of the extendedDsiRNAs of the instant invention were demonstrated across many tissuetypes.

Example 12 In Vivo Efficacy of DsiRNA Agents, Multiple Dose Results

DsiRNA agents possessing DNA duplex extensions were examined for in vivoefficacy of sequence-specific target mRNA inhibition in a repeated doseprotocol at a lower dosage than that of Example 11. Specifically,2′-O-Methyl-modified KRAS-targeting DsiRNA “K249M” (first duplex ofFIGS. 16) and 2′-O-Methyl-modified “right extended” KRAS-targetingDsiRNA “K249D” (second duplex of FIG. 16) were formulated inInvivofectamine™ and injected i.v. in CD1 mice at 2 mg/kg every 3 daysuntil a total of four doses were administered to each mouse. Expressionof KRAS in liver, lung, spleen and kidney tissues was measured 24 hoursafter the final injection was administered (FIGS. 26-29, respectively;each bar presents results obtained for four mice per treatment group),with real-time PCR (RT-PCR) performed in triplicate to assess KRASexpression. Under these conditions, statistically significant reductionsin KRAS levels were observed in liver and spleen tissues of miceadministered the “right-extended” DsiRNA “K249D”. Specific KRAS percentinhibition levels observed in such “K249DNA”-treated tissues andp-values associated with these observations were: liver (46%-90%, mean78%, p=0.002), spleen (36%-80%, mean 62%, P=0.004), kidney (0%, mean 0%,P=0.814**) and lung (17%-38%, mean 26%, P=0.065**). Thus, the in vivoefficacy of the extended DsiRNAs of the instant invention in amulti-dose (low dose) regimen was demonstrated in liver and spleentissues.

Example 13 Further Assessment of In Vivo Efficacy of DsiRNA Agents

Further demonstration of the capability of the extended Dicer substrateagents of the invention to reduce gene expression of specific targetgenes in vivo is performed via administration of the DsiRNAs of theinvention to mice or other mammalian subjects, either systemically(e.g., by i.v. or i.p. injection) or via direct injection of a tissue(e.g., injection of the eye, spinal cord/brain/CNS, etc.). Measurementof additional target RNA levels are performed upon target cells (e.g.,RNA levels in liver and/or kidney cells are assayed following injectionof mice; eye cells are assayed following ophthalmic injection ofsubjects; or spinal cord/brain/CNS cells are assayed following directinjection of same of subjects) by standard methods (e.g., Trizol®preparation (guanidinium thiocyanate-phenol-chloroform) followed byqRT-PCR).

In any such further in vivo experiments, an extended Dicer substrateagent of the invention (e.g., a left-extended or right-extended DsiRNA)can be deemed to be an effective in vivo agent if a statisticallysignificant reduction in RNA levels is observed when administering anextended Dicer substrate agent of the invention, as compared to anappropriate control (e.g., a vehicle alone control, a randomized duplexcontrol, a duplex directed to a different target RNA control, etc.).Generally, if the p-value (e.g., generated via 1 tailed, unpairedT-test) assigned to such comparison is less than 0.05, an extended Dicersubstrate agent (e.g., left-extended or right-extended DsiRNA agent) ofthe invention is deemed to be an effective RNA interference agent.Alternatively, the p-value threshold below which to classify an extendedDicer substrate agent of the invention as an effective RNA interferenceagent can be set, e.g. at 0.01, 0.001, etc., in order to provide morestringent filtering, identify more robust differences, and/or adjust formultiple hypothesis testing, etc. Absolute activity level limits canalso be set to distinguish between effective and non-effective extendedDicer substrate agents. For example, in certain embodiments, aneffective extended Dicer substrate agent of the invention is one thatnot only shows a statistically significant reduction of target RNAlevels in vivo but also exerts, e.g., at least an approximately 10%reduction, approximately 15% reduction, at least approximately 20%reduction, approximately 25% reduction, approximately 30% reduction,etc. in target RNA levels in the tissue or cell that is examined, ascompared to an appropriate control. Further in vivo efficacy testing ofthe extended Dicer substrate agents (e.g., left-extended andright-extended DsiRNA agents) of the invention is thereby performed.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying DsiRNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

What is claimed is:
 1. A double stranded nucleic acid moleculecomprising: a first strand of 26-60 nucleotides in length; a secondstrand of 26-60 nucleotides in length, where said first and secondstrands form a duplex; wherein the first strand comprises a loopadjacent to said duplex; and wherein said molecule comprises adiscontinuity between a terminus of said first strand and a terminus ofsaid second strand.
 2. The double stranded nucleic acid molecule ofclaim 1, wherein the second strand comprises a 5′ phosphate, a 5′phosphonate or a 5′ borophosphonate.
 3. The double stranded nucleic acidmolecule of claim 1, wherein one or both of said first and secondstrands comprise(s) a 5′ phosphate.
 4. The double stranded nucleic acidmolecule of claim 1, wherein the loop comprises a hairpin, a stemloop oran extended loop.
 5. The double stranded nucleic acid molecule of claim1, wherein the loop comprises a tetraloop.
 6. The double strandednucleic acid molecule of claim 1, wherein the loop comprises at leastfour nucleotides that increase the melting temperature of a duplexadjacent to the loop.
 7. The double stranded nucleic acid molecule ofclaim 1, comprising a universal base.
 8. The double stranded nucleicacid molecule of claim 1, comprising an abasic linker.
 9. The doublestranded nucleic acid molecule of claim 5, wherein said tetraloop has anucleic acid sequence selected from the group consisting of UNCG, GNRA,CUUG, d(GNNA), d(GNAB), d(CNNG), and d(TNCG).
 10. The double strandednucleic acid molecule of claim 5, wherein the tetraloop has a nucleicacid sequence selected from the group consisting of UUCG, GAAA, d(GTTA),and d(TTCG).
 11. The double stranded nucleic acid molecule of claim 5,wherein the tetraloop is flanked at the 5′ end by a nucleic acidsequence selected from the group consisting of C, CC, G, and GG.
 12. Thedouble stranded nucleic acid molecule of claim 5, wherein the tetraloopis flanked at the 5′ end by a nucleic acid sequence that duplexes with anucleic sequence selected from the group consisting of C, CC, G, and GG.13. The double stranded nucleic acid molecule of claim 5, wherein thetetraloop is flanked at the 3′ end by a nucleic acid sequence selectedfrom the group consisting of C, CC, G, and GG.
 14. The double strandednucleic acid molecule of claim 5, wherein the tetraloop is flanked atthe 3′ end by a nucleic acid sequence that duplexes with a nucleicsequence selected from the group consisting of C, CC, G, and GG.
 15. Thedouble stranded nucleic acid molecule of claim 1, wherein the loopcomprises residues selected from the group consisting ofribonucleotides, deoxyribonucleotides, modified nucleotides, nucleotideanalogs and combinations thereof.
 16. The double stranded nucleic acidmolecule of claim 1, wherein the double stranded molecule is conjugatedto a non-nucleic acid moiety.
 17. The double stranded nucleic acidmolecule of claim 16, wherein the non-nucleic acid moiety is a peptideor an organic compound.
 18. The double stranded nucleic acid molecule ofclaim 17, wherein the organic compound is a dye
 19. The double strandednucleic acid molecule of claim 17, wherein the organic compound ischolesterol.
 20. The double stranded nucleic acid molecule of claim 1,in combination with a sugar.
 21. The double stranded nucleic acidmolecule of claim 16, wherein the non-nucleic acid moiety increasescellular uptake as compared to a double stranded nucleic acid that isnot conjugated to a non-nucleic acid moiety.
 22. The double strandednucleic acid molecule of claim 16, wherein the non-nucleic acid moietyincreases cellular targeting of the double stranded molecule as comparedto a double stranded nucleic acid that is not conjugated to anon-nucleic acid moiety.
 23. The double stranded nucleic acid moleculeof claim 16, wherein the non-nucleic acid moiety increases stability ofthe double stranded molecule as compared to a double stranded nucleicacid that is not conjugated to a non-nucleic acid moiety.
 24. The doublestranded nucleic acid molecule of claim 16, wherein the non-nucleic acidmoiety facilitates tracking of the double stranded molecule as comparedto a double stranded nucleic acid that is not conjugated to anon-nucleic acid moiety.
 25. The double stranded nucleic acid moleculeof claim 16, wherein the non-nucleic acid moiety increases the bindingaffinity of the double stranded molecule as compared to a doublestranded nucleic acid that is not conjugated to a non-nucleic acidmoiety.
 26. The double stranded nucleic acid molecule of claim 16,wherein the non-nucleic acid moiety decreases the immunogenicity of thedouble stranded molecule as compared to a double stranded nucleic acidthat is not conjugated to a non-nucleic acid moiety.
 27. The doublestranded nucleic acid molecule of claim 16, wherein the non-nucleic acidmoiety improves the pharmacokinetic properties of the double-strandedmolecule as compared to a double stranded nucleic acid that is notconjugated to a non-nucleic acid moiety.
 28. The double stranded nucleicacid molecule of claim 16, wherein the non-nucleic acid moiety improvesthe biodistribution properties of the double-stranded molecule ascompared to a double stranded nucleic acid that is not conjugated to anon-nucleic acid moiety.
 29. The double stranded nucleic acid moleculeof claim 16, wherein the double stranded molecule is conjugated at theloop
 30. The double stranded nucleic acid molecule of claim 1, whereinthe double stranded molecule is nuclease resistant.
 31. The doublestranded nucleic acid molecule of claim 30, wherein the nuclease ispresent in the endosome.
 32. The double stranded nucleic acid moleculeof claim 30, wherein the nuclease is selected from the group consistingof: RNAse III, RNase P, RNase T1, RNase A (a, w, 4, 4/5),oligonucleotidase, XRN1, and XRN2.
 33. The double stranded nucleic acidmolecule of claim 1, wherein the double stranded molecule comprises atleast one deoxyribonucleotide.
 34. The double stranded nucleic acidmolecule of claim 1, wherein the double stranded molecule comprises oneor more modified nucleotides.
 35. The double stranded nucleic acidmolecule of claim 34, wherein the modified nucleotide is aphosphorothioate-modified nucleotide residue (PS-NA).
 36. The doublestranded nucleic acid molecule of claim 34, wherein the modifiednucleotide is a peptide nucleic acid (PNA).
 37. The double strandednucleic acid molecule of claim 34, wherein the modified nucleotidecomprises a modified base and/or a modified sugar moiety.
 38. The doublestranded nucleic acid molecule of claim 34, wherein the modifiednucleotide is selected from the group consisting of:dideoxyribonucleotides, acyclonucleotides 3′-deoxyadenosine(cordycepin), 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine(ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T).
 39. The double strandednucleic acid molecule of claim 37, wherein the modified sugar moiety isselected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy,2′-fluoro, 2′-allyl, 2′-O[2-(methylamino)-2-oxoethyl], 4′-thio,4′-CH2-O-2′-bridge, 4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and2′-O-(N-methylcarbamate).
 40. The double stranded nucleic acid moleculeof claim 34, wherein at least one internucleoside linkage is modified.41. The double stranded nucleic acid molecule of claim 40, wherein theinternucleoside linkage modification is selected from the groupconsisting of: methylphosphonate, phosphorothioate, and phosphotriestermodifications.
 42. The double stranded nucleic acid molecule of claim 1,further comprising a fluorescein.
 43. The double stranded nucleic acidmolecule of claim 34, wherein the modified nucleotide comprises an LNAmodification.
 44. The double stranded nucleic acid molecule of claim 34,wherein the modified nucleotide is selected from the group consisting of2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-0-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge,4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O-(N-methlycarbamate).45. The double stranded nucleic acid molecule of claim 1, wherein saidsecond strand comprises a 3′ overhang.
 46. The double stranded nucleicacid molecule of claim 45, wherein the 3′ overhang comprises at leastone modified nucleotide.
 47. The double stranded nucleic acid moleculeof claim 45, wherein the modified nucleotide of said 3′ overhang is a2′-O-methyl ribonucleotide.
 48. The double stranded nucleic acidmolecule of claim 45, wherein all nucleotides of said 3′ overhang aremodified nucleotides.
 49. The double stranded nucleic acid molecule ofclaim 45, wherein the 3′ overhang is two nucleotides in length andwherein said modified nucleotide of said 3′ overhang is a 2′-O-methylmodified ribonucleotide.
 50. The double stranded nucleic acid moleculeof claim 1, wherein the first strand has a nucleotide sequence that isat least 80%, 90%, 95% or 100% complementary to the second strandnucleotide sequence.
 51. The double stranded nucleic acid molecule ofclaim 1, comprising a phosphate backbone modification selected from thegroup consisting of a phosphonate, a phosphorothioate and aphosphotriester.
 52. The double stranded nucleic acid molecule of claim1, wherein the double stranded molecule reduces target gene expressionin a mammalian cell by an amount (expressed by %) selected from thegroup consisting of at least 10%, at least 50% and at least 80-90%. 53.The double stranded nucleic acid molecule of claim 1, wherein the doublestranded molecule, when introduced into a mammalian cell, reduces targetgene expression by at least 70% when transfected into said cell at aconcentration selected from the group consisting of 1 nM or less, 200 pMor less, 100 pM or less, 50 pM or less, 20 pM or less and 10 pM or less.54. The double stranded nucleic acid molecule of claim 1, wherein atleast 50% of the ribonucleotide residues of said double strandedmolecule are unmodified ribonucleotides.
 55. The double stranded nucleicacid molecule of claim 1, wherein at least 50% of the ribonucleotideresidues of said first strand are unmodified ribonucleotides.
 56. Thedouble stranded nucleic acid molecule of claim 1, wherein at least 50%of the ribonucleotide residues of said second strand are unmodifiedribonucleotides.
 57. The double stranded nucleic acid molecule of claim1, wherein 100% of the ribonucleotide residues of said double strandedmolecule are unmodified ribonucleotides.
 58. The double stranded nucleicacid molecule of claim 1, wherein 100% of the ribonucleotide residues ofsaid first strand are unmodified ribonucleotides.
 59. The doublestranded nucleic acid molecule of claim 1, wherein 100% of theribonucleotide residues of said second strand are unmodifiedribonucleotides.
 60. The double stranded nucleic acid molecule of claim1, wherein at least 50% of the ribonucleotide residues of said doublestranded molecule are modified ribonucleotides.
 61. The double strandednucleic acid molecule of claim 1, wherein 100% of the nucleotideresidues of said double stranded molecule consist of modifiedribonucleotides.
 62. The double stranded nucleic acid molecule of claim1, wherein at least 50% of the residues of said double stranded moleculeare deoxyribonucleotides.
 63. The double stranded nucleic acid moleculeof claim 1, wherein 100% of the nucleotide residues of said doublestranded molecule consist of modified deoxyribonucleotides.
 64. Thedouble stranded nucleic acid molecule of claim 1, wherein the secondoligonucleotide strand, starting from the nucleotide residue of saidsecond strand that is complementary to the 5′ terminal nucleotideresidue of said first oligonucleotide strand and toward the 5′ end ofsaid second strand, comprises alternating modified and unmodifiednucleotide residues.
 65. The double stranded nucleic acid molecule ofclaim 1, wherein the first strand is of a length selected from the groupconsisting of: 25 to 30 nucleotides, 33 to 49 nucleotides, 35 to 49nucleotides and 37 to 49 nucleotides.
 66. The double stranded nucleicacid molecule of claim 1, wherein the second strand possesses a 3′overhang of 1-4 nucleotides in length.
 67. The double stranded nucleicacid molecule of claim 1, wherein the 3′ overhang is 1-3 nucleotides inlength.
 68. The double stranded nucleic acid molecule of claim 67,wherein the 3′ overhang is 1-2 nucleotides in length.
 69. The doublestranded nucleic acid molecule of claim 1, wherein the molecule is anisolated, synthetic or an isolated, synthetic nucleic acid molecule. 70.A method for reducing expression of a target gene in a cell, comprising:contacting a cell with the double stranded nucleic acid molecule ofclaim 1, in an amount effective to reduce expression of a target gene ina cell in comparison to a reference double stranded nucleic acidmolecule.
 71. A method for reducing expression of a target gene in ananimal, comprising: treating an animal with the double stranded nucleicacid molecule of claim 1, in an amount effective to reduce expression ofa target gene in a cell of the animal in comparison to a referencedouble stranded nucleic acid molecule.
 72. The method of claim 70,wherein the double stranded nucleic acid molecule possesses enhancedpharmacokinetics when compared to an appropriate control double strandednucleic acid molecule.
 73. The method of claim 70, wherein the doublestranded nucleic acid molecule possesses enhanced pharmacodynamics whencompared to an appropriate control double stranded nucleic acidmolecule.
 74. The method of claim 70, wherein the double strandednucleic acid molecule possesses reduced toxicity when compared to anappropriate control double stranded nucleic acid molecule.
 75. Themethod of claim 70, wherein the double stranded nucleic acid moleculepossesses enhanced intracellular uptake when compared to an appropriatecontrol double stranded nucleic acid molecule.
 76. A pharmaceuticalcomposition for reducing expression of a target gene in a cell of asubject comprising the double stranded nucleic acid molecule of claim 1,in an amount effective to reduce expression of a target gene in a cellin comparison to a reference double stranded nucleic acid molecule and apharmaceutically acceptable carrier.
 77. A method of synthesizing thedouble stranded nucleic acid molecule of claim 1, comprising chemicallyor enzymatically synthesizing said double stranded molecule.
 78. A kitcomprising the double stranded nucleic acid molecule of claim 1, andinstructions for its use.